JP7005504B2 - Biomolecule immobilization method - Google Patents

Description

Cross-reference to related applications This application claims the priority benefit to European patent application EP16156777.1 filed February 22, 2015. The entire content of this application is incorporated by reference in its entirety.

Background of the invention
1. Technical Field The present invention relates to a method for immobilizing a biomolecule containing at least one sulfhydryl group. The method comprises contacting the modified metal surface with a biomolecule and irradiating the resulting surface with UV rays in the presence of a photoinitiator, wherein the metal surface is a covalently bonded terminal thiol or dithiol to the metal surface. It is modified with a crosslinker compound containing a group, a spacer group holding an isolated double or triple bond at the other end.

2. Advanced Techniques The detection and quantification of analytes present in samples, such as biomolecules and other molecules that affect biological processes, is essential for analytical testing. For example, the detection of biomolecules, which are markers of biological activity or disease, is important for the diagnosis of medical conditions and pathology. However, the conversion of an analyte such as a biomolecule into an available signal is partly the complexity of converting a detection event, eg, an antibody bound to an antigen, into a detectable signal that can be converted into perceptible data. It is difficult because of. Some assays, such as the Enzyme-linked Immunosorbent Assay (ELISA), detect biomolecules by monitoring binding events that produce light or reaction products that result in color changes in the sample. One advantage of these types of assays is that they are extremely sensitive. However, the drawback of these assays, such as ELISA assays, is that they typically require a long time to generate a detectable signal and require multiple steps to complete.

Recently, other methods have been developed that eliminate the complexity and time involved in signal generation while preserving the sensitivity of traditional immunoassays. One strategy is to link the sensitivity of the immunoassay with electrochemical measurements, for example by using highly selective antibodies that have a high affinity for the analyte. By linking the detection event to an electrical signal, information about the presence and concentration of the analyte in the sample can be instantly converted into an electrical signal. Several detection concepts and related devices have been developed over the last few decades. The most common traditional techniques are cyclic voltammetry, chronoamperometry, chronopotencyometry, and impedance spectroscopy.
However, the general performance of electrochemical sensors is often determined by the surface architecture that connects the sensing element to the biological sample at the nanoscale. Electrochemical biosensors have been plagued by a lack of surface architecture that allows for high sufficient sensitivity and unique identification of responses to desired biochemical events.
Therefore, there is a need for an electrochemical biosensor that maintains the desired aspects of an electrochemical sensor, such as easily measurable signals and prospects for miniaturization, while having the high sensitivity of traditional assays such as ELISA assays.

U.S. Patent Application US 2012/0228155 is a method for making a functionalized electrode for detecting a target analyte, which is described below.
A conductive surface, such as a gold electrode, with a mixture of a first thiol compound with a terminal amino group and a second thiol compound with a terminal OH, alkoxy, methyl, sugar, diionic or polar nonionic group. Upon contact, the sulfhydryl groups of the first and second thiol compounds bind to the conductive surface, thereby creating a monolayer on the surface of the conductive surface;
Contacting the monomolecular layer on the surface of the conductive surface with an amine-reactive functional group and a heterobifunctional linker containing a diazirine or maleimide moiety; and specificizing the monomolecular layer on the surface of the conductive surface. The present invention relates to a method comprising contacting with a ligand that binds to a target analyte, thereby making a functionalized electrode for detecting the target analyte.
If the heterobifunctional linker contains sulfo-NHS diazirine (sulfo-SDA), this method further exposes the monolayer on the surface of the conductive surface to UV irradiation, thereby detecting the target analyte. Includes making functionalized electrodes.

U.S. Pat. No. 6,580,571 relates to a method for making a biosensor containing a substrate to which a hydrophilic polymer is attached. This method is the following process:
The step of forming a self-assembled monomolecular film on a substrate (where self-assembly is formed by alkanethiol); a photoradical generator-containing solution is coated on this substrate to give the photoradical generator. A step of allowing the substrate to bind to a self-assembled monomolecular film, a step of coating the substrate with a hydrophilic polymer-containing solution (where the hydrophilic polymer is a polysaccharide having a double bond with a carboxyl group). ) And this substrate are exposed to light to generate reactive groups from a photoradical generator, which comprises covalently bonding the hydrophilic polymer to the reactive group via a double bond of the hydrophilic polymer, whereby the biosensor , Because the hydrophilic polymer contains the substrate attached to it, and the carboxyl group contained in the hydrophilic polymer bonded to the substrate in the biosensor immobilizes the target physiologically active substance on the biosensor. Used for.

Chinese patent application CN 104 597 230 is a method for producing a functionalized polymer film, which is described in the following steps:
The step of forming a terminal-functionalized self-assembling monomolecular layer on the surface of the substrate of the biochip, eg, a terminal-functionalized thiol or dithiol compound bonded to the gold surface; The step of grafting to the ends by chemical bonds; the step of spin-coating the polymer solution on the resulting surface formed; and UV irradiation on the biochip having the spin-coated polymer surface to the polymer grafted on the surface. It suggests a method comprising the step of forming a chemical bond that forms a polymer film under UV light.

Outline of the Invention Therefore, the present invention is a method for immobilizing a biomolecule containing at least one sulfhydryl group.
(a) If necessary, a step of treating the biomolecule with a reducing agent to cleave existing-SS-bonds within the biomolecule, or
(b) If necessary, treat the biomolecule with an acylating agent having a protected sulfhydryl group to deprotect the sulfhydryl group;
(c) Step of contacting the surface of the modified metal with biomolecules;
(d) Including the step of irradiating the resulting surface with UV in the presence of a photoinitiator.
Here, the metal surface is
The terminal thiol or dithiol group covalently bonded to the metal surface is contained, and the terminal thiol or dithiol group is present.
b. With respect to said method, which is connected to a spacer group, wherein the spacer group is modified with a cross-linker compound that retains an isolated CC double bond or CC triple bond at the other end.

Furthermore, the present invention relates to a compound of the following formula (I).
HS- (CH ₂ ) _m -CH (ZH) -SPACER- (CH ₂ ) _p -A (I)
During the ceremony
m is an integer from 2 to 6
A is selected from -CH = CH ₂ and -C≡CH, and
Z is S or a single bond,
SPACER is the following formula
-(CH ₂ ) _n- (C = O) _x -Y-(CH ₂ CH ₂ -O) _y- (CH ₂ ) _r- (C = O) _v -X-
Is the basis of
here,
X and Y are independently NH or O, respectively.
n is 0 or an integer from 1 to 10 and
x and v are 0 or 1 independently, respectively.
y is an integer from 1 to 20
r and p are independently selected from integers 1 to 6.

Another aspect of the present invention is an intermediate of formula (II) below.

In the equation, A, SPACER, m and s have the meaning given for equation (I).
Furthermore, the present invention relates to a modified metal surface, wherein at least one of the metal atoms on the surface is covalently bonded to at least one thiol group of the compound of formula (I) of the present invention.
The final aspect of the present invention is a kit for performing a method for immobilizing a biomolecule according to the present invention, and is described below.
(i) A substrate having a modified metal surface of the present invention,
(ii) Optional storage unit containing the appropriate reducing agent, or
(iii) An optional storage unit containing a suitable acylating agent with a protected sulfhydryl group and a deprotecting agent for the sulfhydryl group.
(iv) Storage units containing suitable photoinitiators, and
(v) A kit containing a pamphlet explaining the conditions for performing this method.

FIG. 3 shows a fluorescence photograph of the complementary metal oxide semiconductor (CMOS) chip of the present invention under different conditions compared to a surface modified with lipoamide-PEG (11) -maleimide. A fluorescence photograph of the CMOS chip of the present invention in which a monoclonal mouse antibody is immobilized is shown. A fluorescent photograph of the CMOS chip of the present invention in which a polyclonal rabbit anti-ACTH antibody is immobilized is shown. The schematic diagram of the immobilization of the biomolecule of this invention is shown.

Detailed description of the invention
I. Term List Unless otherwise specified, technical terms are used according to customary usage. Definitions of general terms in molecular biology are Benjamin Lewin, Genes VII (Oxford University Press Publishing), 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (B1ackwel1 Publishers Publishing), 1994 ( ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, (Wiley, John & Sons, Inc.), 1995 (ISBN 0471186341); and other similar references. Be done.

As used herein, the singular terms "a", "an", and "the" include multiple references unless the context explicitly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context explicitly indicates otherwise. Also, as used herein, the term "comprises" means "includes". Thus, "including A or B" means including A, B, or A and B. Furthermore, it should be understood that all nucleotide or amino acid sizes, as well as all molecular weight or molecular weight values given to nucleic acids or polypeptides or other compounds, are approximate and are provided for illustration purposes. Methods and materials similar to or equivalent to those described in the present invention can be used for carrying out or testing the present disclosure, but appropriate methods and materials will be described below. In the event of a contradiction, the present specification, including explanations of terms, will prevail. Moreover, the materials, methods, and examples are merely exemplary and are not intended to be limiting.

To facilitate scrutiny of the various examples of the present disclosure, the following description of special terms is provided.
Biomolecule: A bioactive molecule that can be derived from a biological source or produced synthetically.
Allergen: A non-parasitic antigen that can stimulate type I hypersensitivity reactions. Type I allergy is an immunoglobulin E (IgE) antibody against an otherwise harmless antigen called an allergen that can originate from multiple sources of allergens (eg, mites, pollen, animals, insects, molds, and food). Production. IgE-mediated presentation of the allergen to T cells results in T cell activation and chronic allergic inflammation (eg, chronic asthma, atopic dermatitis), especially after repeated contact with the allergen. This event also induces an increase in allergen-specific serum IgE levels and patients. Common allergens include: trees and other plant-derived allergens, such as the Betula verrucosa allergens Bet v I, Bet v 2, and Bet v 4; the Juniperous oxycedrus allergen Jun. o 2; Castanea sativa allergen Cas s 2; and Hevea brasiliensis allergen Hev b I, Hev b 3, Hev b 8, Hev b 9, Hev b 10 and Hev b 11; Grasses such as Phleum pretense allergens Phl p I, Phl p 2, Phl p 4, Phl p Sa, Phlp 5, Phlp 6, Phlp 7, Phl p 11 and Phl p 12; Parietaria judaica allergen Par j 2.01011; and Artemisia vulgaris allergens Art v I and Art v 3; mites such as Dermatophagoides pteronyssinus allergens Der p I, Der p 2. Der p 5, Der p 7, Der p 8, and Der p 10; Tyrophagu putrescentiae allergen Tyr p 2; Lepidoglyphus destrtuctor allergens Lep d 2.01 and Lep d 13; Euroglyphus maynei allergen Euro 2.0101; animals, eg cats, eg Felis domesticus allergen Feld I; Penaeus aztecus allergen Pen a I; Cyprinus carpo allergen I; and as well as albumins from cats, dogs, cows, mice, rats, pigs, sheep, chickens, rabbits, hamsters, horses, pigeons, and guinea pigs; fungi such as penicillium citrinum (Pen). icillium citrinum) allergens Pen c 3 and Pen c 19; Penicillium notatum allergens Penn 13; Aspergillus fumigatus allergens Asp f I, Asp f3, Asp f4, Asp f6, Asp f7 Lunaria alternata allergens Alt a I and Alt a 5; Malassezia furfur allergens Mal f I, Mal f 5, Mal f 6, Mal f 7, Mal f 8 and Mal f 9; Insects , For example, the Blatella germanica allergens Bla g 2, Bla g 4, and Bla g 5; the Apis mellifera allergens Api m 2 and Api m I; the Vespula vulgaris allergen Ves v. 5; Vespula germanica allergen Ves g 5; and Polstes annularis allergen Pol a 5; foods such as Malus domestica allergens Mal d I and Mal d 2; Apium graveolens) allergens Api g I and Api g 1.0201; Daucus carota allergens Dau c I; and Arachis hypogaea allergens Ara h 2 and Ara h 5. In some embodiments, the allergen or portion thereof is part of a functionalized surface or electrode, and thus the disclosed functionalized surface is used to measure the presence and concentration of antibody in the sample that specifically binds to the allergen. can do. In some embodiments, the antibody that specifically binds to the allergen or portion thereof is part of the disclosed functionalized surface or electrode, and thus the disclosed functionalized electrode can be used to measure the presence and concentration of the allergen. ..

An "antibody" is an immunoglobulin or immunoglobulin-like substance that collectively eliminates binding to other molecules and specifically binds to the molecule of interest (or a group of very similar molecules of interest). Molecules (eg, but not limited to, IgA, IgD, IgE, IgG and IgM, including combinations thereof), as well as any vertebrates such as vertebrates such as mammals such as humans, goats, rabbits. And similar molecules produced during immune reactions in mice and the like and fragments thereof. An "antibody" typically comprises a polypeptide ligand having at least one light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Exemplary antibodies include polyclonal and monoclonal antibodies.
Term Antibodies also include paratope sequences that can bind to other analytes.

Immunoglobulins are composed of heavy chains and light chains, and have variable regions called variable heavy chain (VH) regions and variable light chain (VL) regions, respectively. Together, the VH and VL regions are involved in binding to antigens recognized by immunoglobulins. Exemplary immunoglobulin fragments include, but are not limited to, proteolytic immunoglobulin fragments (eg, technically well known F (ab') 2 fragments, Fab'fragments, Fab'-SH fragments and Fab fragments). Recombinant immunoglobulin fragments (eg, sFv fragment, dsFv fragment, bispecific sFv fragment, bispecific dsFv fragment, F (ab) '2 fragment), single chain Fv protein ("scFv"), and disulfide stabilization. Examples include Fv proteins (“dsFv”). Other examples of antibodies include diabodies and triabodies (technically known), as well as camelid antibodies. "Antibodies" also include genetically engineered molecules such as chimeric antibodies. "Antibodies" also include genetically engineered antibodies such as chimeric antibodies (eg humanized mouse antibodies) and heterobound antibodies (eg bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

Each heavy and light chain contains a constant region and a variable region (regions are also known as "domains"). Combined, the heavy and light chain variable regions bind specifically to the antigen. The light and heavy chain variable regions contain "framework" regions interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". The framework area and the scope of the CDRs are defined (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, Public Health Service, National Institutes of Health, incorporated by reference. See Health, Bethesda, Md. (NIH Publication No. 91-3242)). The Kabat database is currently maintained online. Within the species, the sequences of various light or heavy chain framework regions are relatively conserved. The framework region of the antibody, which is the framework region where the light chain and the heavy chain are combined, works to position and align the CDR in the three-dimensional space, and holds the CDR in an orientation suitable for antigen binding, for example.
CDRs are primarily involved in the binding of antigens to epitopes. The CDRs of each strand are typically numbered sequentially from the N-terminus and are referred to as CDR1, CDR2 and CDR3, and the particular CDR is typically also identified by the strand in which it is located. Thus, VH CDR3 is located within the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is the CDRI of the variable domain of the light chain of the antibody in which it is found.

A "monochromal antibody" is an antibody produced by a single clone of a B lymphocyte or by a cell into which the light and heavy chain genes of the single antibody have been transfected or transduced. Monoclonal antibodies are produced by methods well known to those of skill in the art, such as the production of hybredoma antibody-forming cells from fusion of myeloma cells and immunospleen cells. These fused cells and their progeny are called "hybridomas". Monoclonal antibodies include humanized monoclonal antibodies. [0048] A "humanized" immunoglobulin is an immunoglobulin containing one or more CDRs from the human framework region and non-human (eg, mouse, rat or synthetic) immunoglobulins. The non-human immunoglobulins that form the CDRs are called "donors" and the human immunoglobulins that form the framework are called "acceptors". In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, eg, at least about 85-90%, eg, about 95% or more. Thus, perhaps all but the CDRs of humanized immunoglobulin are substantially identical to a significant portion of the native human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody that forms the CDR. The acceptor framework for humanized immunoglobulins or antibodies can have a limited number of substitutions with amino acids obtained from the donor framework. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed using genetic engineering (see, eg, US Pat. No. 5,585,089).

In some embodiments, the antibody is, for example, an antigen that is part of a disclosed functionalized electrode covalently attached to a functionalized thiol or dithiol compound that is covalently attached to a thiol or dithiol compound or a functionalized thiol or dithiol compound that itself is attached to the electrode surface. It specifically binds to the antigen of interest. In some embodiments, the antigen-specific antibody of interest is, for example, one of the disclosed functionalized electrodes covalently attached to a thiol or dithiol compound or a functionalized thiol or dithiol compound that itself is attached to the electrode surface. It is a department. In some embodiments, the antibody is part of a detection reagent comprising an enzyme.

Antigen: A compound, composition, or substance that can be specifically bound by a specific product of humoral or cell-mediated immunity, such as an antibody molecule or T cell receptor. The antigen can be any type of molecule, eg, haptens, simple intermediate metabolites, sugars (eg oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (eg polysaccharides), phosphorus. Examples include lipids, nucleic acids and proteins. Antigens of the general classification include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasite antigens, tumor antigens, autoimmune diseases, allergic reactions and antigens involved in transplant rejection. Examples include toxins, as well as other technically well-known antigens.
In some embodiments, the antigen is an object, such as an antibody that is part of a disclosed functionalized electrode covalently attached to a functionalized thiol or dithiol compound that is covalently attached to, for example, a thiol or dithiol compound or a functionalized thiol or dithiol compound that itself is attached to the electrode surface. It is a ligand for the antibody. In some embodiments, the antigen of interest is, for example, part of a disclosed functionalized electrode covalently attached to, for example, a thiol or dithiol compound or a functionalized thiol or dithiol compound that itself is attached to the electrode surface.

Aptamer: A small nucleic acid and peptide molecule that specifically binds to a target molecule such as a target biomolecule, for example, an analyte such as a target analyte. In some embodiments, the aptamer is part of the disclosed modified surface, such as a functionalized electrode.
Bacterial pathogens: Bacteria that cause disease (pathogenic bacteria). Disclosure Examples of pathogenic bacteria from which the antigen used for the functionalized electrode can be derived include, but are not limited to, Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, among others. Actinomyces sp. (eg Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (Eg Aeromonas hydrophila, Aeromonas hydrophila) Biovar sobria (Aeromonas veronii biovar sobria), and Aeromonas caviae, Anaplasma phagocytophilum, Alcaligenes xy los Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (eg Bacillus anthracis, Bacillus anthracis, Bacillus cereus) Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus, Bacteroides sp. (eg Bacteroides fragilis), Bartonella ) (For example, Bartonella bacilliformis and Bartonella henselae), Bifid bacterium species (Bifid) obacterium sp.), Bordetella sp. (eg Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Bordetella sp. (eg Borrelia sp.) -Borrelia recurrentis, and Bordetella burgdorferi, Brucella sp. (eg Brucella abortus, Brucella canis, Brucella melintensis) ) And Brucella suis), Bordetella sp. (Eg Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. ) (For example, Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardio Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp., Citrobacter sp. Corynebacterium sp. (For example, Corynebacterium diphther) iae), Corynebacterium jeikeum and Corynebacterium, Clostridium sp. (eg Clostridium perfringens, Clostridium docile, Clostridium docile) Clostridium botulinum and Clostridium tetani, Eikenella corrodens, Enterobacter sp. (eg Enterobacter aerogenes, Enterobacter aerogenes) , Enterobacter cloacae and Escherichia coli (Escherichia coli, opportunistic Escherichia coli, eg enterotoxogenic E. coli, intestinal invasive Escherichia coli, intestinal pathogenic Escherichia coli, intestinal hemorrhagic Escherichia coli, including intestinal flocculant Escherichia coli and urinary tract pathogenic Escherichia coli), Enterococcus sp. (eg Enterococcus faecalis and Enterococcus faecium), Ehrlichia sp. For example, Ehrlichia chafeensia and Ehrlichia canis, Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Francisella tularensis, for example. Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (Example) Haemophilus injluenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainjluenzae, Haemophilus parainjluenzae, haemophilus parainjluenzae, haemophilus parainjluenzae, haemophilus parainjluenzae )).
In addition, Helicobacter sp. (eg Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Eingella kingii, Elebsiella sp. ) (For example, Elebsiella pneumoniae, Elebsiella granulomatis and Elebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes. Logans (Leptospira interrogans), Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Moraxella catarrhalis, Moraxella catarrhalis ), Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (For example, Mycobacterium leprae, Mycobacterium tuberculosis) ), Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum, Mycoplasm species sp.) (For example, Mycoplasma pneumoniae, Mycoplasma hominis (M) ycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (eg Nocardia asteroides), Nocardia cyriacigeorgica and Nocardia brasiliensis )), Neisseria sp. (eg Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides, Species (Prevotella sp.), Porphyromonas sp., Prevotella melami nogenica, Proteus sp. (eg Proteus vulgaris and Proteus vulgaris) Proteus mirabilis)), Providencia sp. (eg, Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas stuartii, Pseudomonas Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (eg Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazeki) , Orientia tsutsugamushi (formerly: Rikecchia tsutsugamushi (R) ickettsia tsutsugamushi)) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and salmonella cholerasuis and salmonella cholerasuis ), Serratia sp. (eg Serratia marescens and Serratia liquifaciens), Shigella sp. (Eg Shigella dysenteriae), Shigera・ Flexneri (Shigella flexneri), Shigella boydii and Shigella sonnei), Staphylococcus sp. (For example, Staphylococcus aureus, Staphylococcus aureus) Epidermidis (Staphylococcus epidermidis), Staphylococcus hemolyticus, Staphylococcus saprophyticus, Streptococcus sp. Ramphenicol-resistant Serratia marcescens, Stenotrophomonas resistant Serratia marcescens, Stenotrophomonas resistant Serratia marcescens, Streptomycin-resistant Serratia marcescens 9V lungs Streptococcus, erythromycin-resistant serotype 14 pneumococcus, opthin-resistant serotype 14 pneumoniae, riphanpicin-resistant serotype 18C pneumococcus, tetracycline-resistant serotype 19F pneumococcus, penicillin-resistant serotype 19F pneumococcus, and trimetoprim-resistant serotype 23F Resistant Streptococcus, Spectinomycin-Resistant Serotype 6B Streptococcus, Streptococcus-Resistant Serotype 9V Streptococcus, Opthin-Resistant Serotype 14 Pneumococcus, Riphanpicin-Resistant Serotype 18C Pneumococcus, Penicillin-Resistant Serotype 19F Pneumococcus, or Trimethoprim-Resistant Streptococcus 23 ), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A Streptococcus (Streptococcus), Streptococcus (Streptococcus), Streptococcus (Streptococcus), Streptococcus (Streptococcus) , Streptococcus agalactiae, Group C Streptococcus, Streptococcus anginosus, Streptococcus equismilis, Group D Streptococcus, Streptococcus bovis Streptococcus anginosus Group G Streptococcus, Spirillum minus, Streptobacillus moniliformi, Treponema sp. (Treponema petenue), Treponema pallidum and Treponema endemicum, Trophyrima whippelii), Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (eg Vibrio cholerae, Vibrio parahemolyticus, Vibrio barnificus) vulnificus), Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio mimicus, Vibrio fimicus, Vibrio holisa , Vibrio metchnilrovii, Vibrio damsela and Vibrio furnisii, Yersinia sp. (eg Yersinia enterocolitica) pestis), and one or more (or a combination thereof) of Yersinia pseudotuberculosis and Xanthomonas maltophilia.

Bacterial antigens suitable for use in the disclosed methods and compositions include proteins that can be isolated, purified or induced from bacteria, polysaccharides, lipopolysaccharides, and adventitial vesicles. In addition, bacterial antigens include lytic solutions and inactivated bacterial preparations. Bacterial antigens can be produced by recombinant expression. Bacterial antigens preferably include epitopes exposed on the surface of the bacterium during at least one step of its life cycle. Bacterial antigens include, but are not limited to, antigens derived from one or more of the specific antigen examples specified below as well as the above-mentioned bacteria.
Gonorrhea (Neiserria gonorrhoeae) antigens include Por (or porn) proteins, such as PorB (see, eg, Zhu et al. (2004) Vaccine 22: 660-669), transferring binding protein, For example, TbpA and TbpB (see, eg, Price et al. (2004) Infect. Immun. 71 (1): 277-283), opacity protein (eg, Opa), reduction-modifiable protein. (Rmp), and outer membrane vesicle (OMV) formulations (eg, Plante et al. (2000) J. Infect. Dis. 182: 848-855; WO 99/24578; WO 99/36544; all incorporated by reference. WO 99/57280; and WO 02/079243).
Chlamydia trachomatis antigens include cellotypes A, B, Ba and C (pathogens of trachoma, causes of blindness), cellotypes Li, L3 (related to inguinal lymphogranulomatosis), and cellotypes D to K. Is done. Chlamydia trachomas antigens also include those identified in WO 00/37494; WO 03/049762; WO 03/068811; and WO 05/002619 (all incorporated by reference), PepA (CT045). ), LcrE (CT089), Art (CT381), DnaK (CT396), CT398, OmpH (CT242), L7 / L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), MurG (CT761), CT396 and CT761, and specific combinations of these antigens.
Treponema pallidum (Treponema pallidum) (Treponema pallidum) Antigen includes TmpA antigen.

The compositions of the present disclosure may comprise one or more antigens derived from a sexually transmitted disease (STD). The antigen can provide prevention or treatment of STDs such as chlamydia, genital herpes, hepatitis (eg HCV), genital warts, gonorrhea, syphilis and / or chancroid (see WO 00/15 255 incorporated by reference). ). The antigen can be from one or more viral or bacterial STDs. The viral STD antigen used in the present invention can be derived from, for example, HIV, herpes simplex virus (HSV-I and HSV-2), human papillomavirus (HPV), and hepatitis virus (HCV). Bacterial STD antigens used in the present invention include, for example, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi, E. coli, and Streptococcus aureus. -It can be derived from Agalactia (Streptococcus agalactiae).
In some embodiments, the disclosed functionalized surface or electrode comprises one or more antigens from one or more of the above organisms. In some embodiments, an antibody that specifically binds to an antigen from one or more of the above organisms is part of the disclosed functionalized electrode, thus in some examples to detect the antigen in the sample. Can be used, for example, to diagnose a particular bacterial infection.

Binding affinity: The affinity of a specific binding factor for a target, for example, the affinity of an antibody for an antigen, the affinity of an antibody for a target analyte such as a target analyte. In one embodiment, the affinity is calculated by the Scatchard method described by Frankel et al., Mo /. Immunol., 16: 101-106, 1979. In another embodiment, the binding affinity is measured by the specific binding agent receptor dissociation rate. In yet another embodiment, high binding affinity is measured by a competitive radioimmunoassay. In some examples, the high binding affinity (K _D ) is at least about 1 × 10 ^-8 M. In other embodiments, the high binding affinity is at least about 1.5 × 10 ^-8 , at least about 2.0 × 10 ^-8 , at least about 2.5 × 10 ^-8 , at least about 3.0 × 10 ^-8 , and at least about 3.5 × 10 ^-8 . , At least about 4.0 × 10 ^-8 , at least about 4.5 × 10 ^-8 or at least about 5.0 × 10 ^-8 M.

Biomolecules: Any molecule of biological origin, but not limited to, synthetic or naturally occurring proteins, glycoproteins, lipoproteins, amino acids, nucleosides, nucleotides, nucleic acids, oligonucleotides, DNA, PNAs, RNA, Examples include carbohydrates, sugars, lipids, fatty acids, haptens, antibiotics, vitamins, enterotoxins and the like. In some examples, a biomolecule is a targeted analyte whose presence and / or concentration or amount can be determined. In some embodiments, the biomolecule is covalently attached to a thiol or dithiol compound and / or a crosslinker, eg, a disclosed functionalized metal surface, particularly a thiol or dithir compound that is part of an electrode.

Chemokines: Classified according to shared structural properties such as the presence of four cysteine residues at storage locations that are key to the formation of small sizes (about 8-10 kilodaltons (kDa) by mass) and their three-dimensional shape. protein. These proteins exert their biological effects by interacting with G protein-binding transmembrane receptors called chemokine receptors that are selectively present on the surface of their target cells. Chemokines are chemokine receptor ligands because they bind to chemokine receptors.
Examples of chemokines include CCL chemokines such as CCL1, CCL2, CCL3, CCL4, CCLS, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20. CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27 and CCL28; CXCL chemokines such as CXCL1, CXCL2, CXCL3, CXCL4, CXCLS, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 There are CXCL16 and CXCL17; XCL chemokines such as XCL1 and XCL2; and CX3CL chemokines such as CX3CL1. In some embodiments, the chemokine or portion thereof is part of the disclosed functionalized electrode. In some embodiments, an antibody that specifically binds to the chemokine or portion thereof is part of a functionalized electrode, so in some examples it can be used to detect the chemokine in a sample. ..

Mobulation, ligation, binding or chaining: chemical coupling of the first unit to the second unit. This includes, but is not limited to, covalent bonds of one molecule to another molecule, non-covalent bonds of one molecule to another molecule (eg, electrostatic bonds), one molecule by hydrogen bond. Includes non-covalent binding of one molecule to another molecule, non-covalent binding of one molecule to another molecule by van der Waals forces, and any combination of such couplings. In some embodiments, the ligand for the target analyte is covalently attached to a thiol or dithiol compound and / or is a cross-linker.
Contact: Place in direct physical association, including both solid and liquid forms.
Control: Standard sample. In some examples, the control can be a known value indicating a known concentration or amount of a target analyte, eg, an analyte such as a biomolecule of interest. In some examples, the functionalized electrode can be calibrated with a known concentration or amount of control, or a set of controls.
The difference between the test sample and the control can be an increase or conversely a decrease. Differences can be qualitative or quantitative differences, eg, statistically significant differences. In some examples, the difference is at least about 10%, eg at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, relative to the control. At least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, Or an increase or decrease of more than 500%.

Complex (complex type): Two proteins, or fragments or derivatives thereof, one protein (or fragment or derivative) and non-protein compounds, molecules or any two or more compounds can be measured in a specific manner. It is said that when they meet with each other, they form a complex. In some examples, the complex is a complex formed between the functionalized electrode and the target analyte.
Covalent bond: An interatomic bond between two atoms, characterized in that these atoms share one or more electron pairs. The term "covalently bound" or "covalently linked" means that two separate molecules are combined into one adjacent molecule, eg, a ligand and thiol specific for a target analyte. Alternatively, the dithiol compound can be covalently attached (eg, directly or indirectly via a linker).
Crosslinker: Homo or homozygous with at least two non-identical groups that are reactive with at least one functional group present in the biomolecule, eg, a sulfhydryl group in a photochemical reaction and another functional group that forms a covalent bond to a metal surface. Heteropolyfunctional reagent. Both functional groups are, in principle, separated from each other by a spacer group. In some examples, the protein crosslinker is sulfhydryl reactive, which protects a sulfhydryl group, eg, a sulfhydryl group present in a biomolecule, eg, a sulfhydryl group present on a cysteine residue, or, eg, an amine group. It means that a covalent bond can be formed with the introduced sulfhydryl group by deprotecting after reacting with a drug having a sulfhydryl group.

Cytokine: A generic name for a variety of soluble proteins and peptides that act as humoral regulators at nano to picomolar concentrations and regulate the functional activity of individual cells and tissues under either normal or pathological conditions. These proteins also directly mediate cell-cell interactions and control processes that occur in the extracellular environment. Cytokines include both naturally occurring peptides and variants that retain full or partial biological activity. Cytokines are cytokine receptor ligands because they bind to cytokine receptors.
Examples of cytokines include interleukins such as IL-1a, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10 and IL-12; interferons such as IFN. -a, IFN- (3 and IFN-y; tumor necrosis factors such as TNF-a and TNF- (3 macrophages; inflammatory proteins such as MIP-1a and MIP-13;) and transforming growth factors such as TGF-( 3. In some examples, the cytokine or portion thereof is part of the disclosed functionalized electrode. In some embodiments, an antibody that specifically binds to the cytokine or portion thereof is the disclosed functionalized electrode. Partial and therefore disclosed functionalized electrodes can be used to determine the presence of cytokines in the sample.

Cyclic voltammetry: An electrochemical technique that can be used to obtain information about the redox potential of an analyte solution or enzyme substrate pair, eg, to select an enzyme substrate pair to include in a disclosed biosensor. It sweeps the voltage between the two values at a fixed speed, but when the voltage reaches V2, it reverses the scan and sweeps the voltage back to V1. The voltage is measured between the reference electrode and the working electrode, while the current is measured between the working electrode and the counter electrode. The obtained measurements are plotted as current vs. voltage. This is also known as a voltammogram. As the voltage rises towards the electrochemical reduction potential of the analyte, so does the current. When this reduction potential was passed while increasing the voltage toward V2, the current dropped and a peak was formed from the time when the oxidation potential was exceeded. As the voltage is reversed and the scan towards V1 is completed, the reaction begins to oxidize the product from the initial reaction again. This results in an increase in non-polar current compared to the forward scan, but as the voltage scan continues towards V1, the current drops again to form a second peak. Reverse scanning also provides information about the reversibility of the reaction at a given scanning rate. The shape of the voltamogram for a given compound depends not only on the scanning rate and the different electrode surfaces after each adsorption step, but also on the catalyst concentration.

Detection: Determining the presence or absence of an agent (eg, a signal or target analyte). In some examples, this may further include quantification. In some examples, electromagnetic signals are used to detect the presence, amount or concentration of an agent, such as an analyte. In some examples, detection is indirect, for example using an enzyme that catalyzes the generation of a detectable signal in the presence of an analyte. In another example, the presence of the analyte reduces the signal, so that the increased concentration of the analyte reduces the signal.
Dithiol group: A cross-linker end group representing, in principle, two thiol or sulfhydryl groups separated by a C _1-5 alkylenediyl group. Most preferably, dithiol can be obtained by reducing the lipoic acid derivative.

Epitope: Antigen determinant. These are specific chemical groups or adjacent or non-adjacent peptide sequences on the molecule that are antigenic, i.e., elicit a specific immune response. Antibodies bind to specific antigenic epitopes based on the three-dimensional structure of the antibody and matching (or cognate) epitopes.
Electromagnetic radiation: A series of electromagnetic waves propagated by simultaneous periodic fluctuations in the intensity of electric and magnetic fields, including radio waves, infrared rays, visible light, ultraviolet (UV) light, X-rays, and gamma rays. In certain examples, electromagnetics are in the form of electrons that can be detected as current changes on the surface of the electrode, eg, the functionalized metal disclosed herein.

Fungal pathogens: Fungi that cause disease. Examples of fungal pathogens used according to the disclosure method and composition are, but are not limited to, Trichophyton rubrum, T. mentagrophytes, Epidermophyton.・ Epidermophyton floccosum, Microsporum canis, Pityrosporum orbiculare (Malassezia furfur), Candida sp. (For example, Candida albican) Aspergillus sp. (eg Aspergillus fumigatus, Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (Eg Cryptococcus sp.) neoformans, Ciyptococcus gattii, Ciyptococcus laurentii and Ciyptococcus albidus, Histoplasma sp. (Pneumocystis sp.) (Eg Pneumocystis jirovecii), and any one or more (or any combination thereof) of the genus Stachybotrys (eg Stachybotrys chartarum). In some embodiments, the disclosed functionalized substrate or electrode comprises one or more antigens from one or more of the above organisms. In some embodiments, the antibody that specifically binds to one or more biological antigens described above is part of a disclosed functionalized electrode, so in some embodiments, for example, a particular fungal infection is diagnosed. The antibody can be used to detect the presence of the antigen in the sample or the fungus in the environmental sample.

Growth factor: A protein that can stimulate cell proliferation and cell differentiation. Examples of growth factors include transformed growth factor β (TGF- (3), granulocyte colony stimulator (G-CSF), granulocyte macrophage colony stimulator (GM-CSF), nerve growth factor (NGF), neuro. Trophin, platelet-derived growth factor (PDGF), erythropoetin (EPO), thrombopoetin (TPO), myostatin (GDF-8), growth differentiation factor-9 (GDF-9, basic fibroblast growth factor (bFGF or FGF2)) , Epithelial growth factor (EGF), hepatocellular growth factor (HGF), etc. In some embodiments, the growth factor or portion thereof is part of the disclosed functionalized electrode. In some embodiments, An antibody that specifically binds to a growth factor or portion thereof is part of the disclosed functionalized electrode, so in some examples the antibody can be used to detect the growth factor in a sample.
Heterogeneous: With respect to molecules such as linkers, "heterologous" usually refers to molecules that are not related to each other, for example as a single molecule. Thus, a "heterologous" linker is one attached to another molecule, which is usually not found naturally, eg, associated with wild-type molecules.
High-throughput technology: Through this process, the analyte present in one sample or multiple samples can be easily identified. In certain cases, modern robotics, data processing and control software, liquid handling equipment, and sensitive detectors are used in combination, so high-throughput technology can be used for short periods of time, eg, using the assays and compositions disclosed herein. Allows rapid detection and / or quantification of the analyte.

Hormones: A classification of small molecules that carry a signal from one cell (or group of cells) to another (or group of cells). Examples of hormones include, among others, amine-tryptophan, such as melatonin (n-acetyl-5-methoxytryptamine) and serotonin; amine-tyrosine, such as tyrosin (thyroid hormone), tri-iodotyronin (thyroid hormone), epinephrine (adrenaline). , Norepinephrine (noradrenaline) and dopamine; peptide hormones such as anti-Murelian hormone (Murelian inhibitor), adiponectin, corticostimulatory hormone (orticotropin), angiotensinogen and angiotensin, antidiuretic hormone (basopressin, arginine vasopressin), atrial Sodium diuretic peptide atriopeptin), calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoetin, holicle stimulating hormone, gastrin, grelin, glucagon, gonadotropin-releasing hormone, growth factor-releasing hormone, human chorionic gonadotropin, human placenta lactogen, growth Hormones, inhibin, insulin, insulin-like growth factor (somatomedin), leptin, luteinizing hormone, melanocite stimulating hormone, oxytocin, parathyroid hormone, prolactin, reluxin, secretin, somatostatin, thrombopoetin, thyroid stimulating hormone and tyrotropin-releasing hormone; Cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstendione, dihydrotestosterone, estradiol, estron, estriol, progesterone and carcitriol (vitamin d3); and ecosanoids such as prostaglandin, leukotriene, prostacycline and thromboxane Can be mentioned. In some embodiments, the hormone or portion thereof is part of the disclosed functionalized electrode. In some embodiments, an antibody that specifically binds to the hormone or portion thereof is part of the disclosed functionalized electrode. Thus, in some examples, the disclosed functionalized electrodes can be used to detect the hormone as well as its precursors and analogs.

Isolated: The "isolated" biocomponent (eg, biomolecule) is substantially separated or purified from the other components in the mixture.
Lagent: 10 kilos that specifically binds to any molecule that specifically binds to the analyte of interest (eg, the target analyte), such as an antibody, protein, peptide or small molecule (eg, the target analyte). Molecules with a molecular mass less than Dalton (kDa)).
Linker and cross-linker: A compound or component that acts as a molecular bridge to operably link two different molecules. In this case, one portion of the linker is operably linked to the first molecule and another portion of the linker is operably linked to the second molecule. Two different molecules can be linked to the linker in a step-by-step manner. The linker has no specific size or content limitation as long as it can serve its purpose as a molecular bridge. Those skilled in the art will appreciate that the linker includes, but is not limited to, chemical chains, compounds, carbohydrate chains, peptides, haptens and the like. Linkers can include, but are not limited to, homobifunctional and heterobifunctional linkers. Heterobifunctional linkers well known to those of skill in the art have one end having a first reactive functional group for specifically binding to a first molecule and a second reactivity for specifically binding to a second molecule. Contains the opposite end with a functional group. Depending on factors such as the molecule to be bound and the conditions under which the detection method is performed, the linker may be length and composition to optimize properties such as flexibility, stability and specific chemical and / or temperature parameters. Can fluctuate.

Nucleic acid: Nucleotide units linked by phosphodiester bonds (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and non-naturally occurring synthetic analogs or combinations thereof), related naturally occurring structural variants. And polymers composed of synthetic analogs of non-natural origin. Thus, this term refers to nucleotides and constitutive analogs of non-natural origin, eg, but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-. Nucleic acid polymers containing O-methylribonucleotides, peptide nucleic acids (PNAs), etc. are included. The polynucleotide can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally less than 50 nucleotides. When a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), it also includes an RNA sequence in which "U" replaces "T" (ie, A, U, G, C). It will be understood that it is included.

Nucleotide sequences are described herein using conventional notation: the left end of a single-stranded nucleotide sequence is the 5'end; the right direction of the double-stranded nucleotide sequence is referred to as the 5'direction. The addition of nucleotides in the 5'to 3'direction to the nascent RNA transcript is called the transcription direction. A DNA strand that has the same sequence as the mRNA is called a "coding strand"; a sequence on the DNA strand that has the same sequence as the mRNA transcribed from the DNA and is located at the 5'to 5'end of the RNA transcript. Is called the "upstream sequence"; the sequence having the same sequence as the RNA and at the 3'to 3'end of the coding RNA transcript is called the "downstream sequence".
"Recombinant nucleic acid" refers to a nucleic acid having a nucleotide sequence that is not naturally bound. This includes nucleic acid vectors containing amplified or organized nucleic acids that can be used to transform suitable host cells. Host cells containing recombinant nucleic acid are referred to as "recombinant host cells". The gene is expressed in this recombinant host cell to produce, for example, a "recombinant polypeptide". Recombinant nucleic acids can also perform non-coding functions (eg, promoters, origins of replication, ribosome binding sites, etc.).

For sequence comparison of nucleic acid sequences, a sequence typically acts as a reference sequence and is compared with the test sequence. When using the sequence comparison algorithm, enter the test and reference sequences into the computer, specify the sequence coordinates, and optionally specify the sequence algorithm program parameters. Use default program parameters. Sequence alignment methods for comparison are technically well known. Optimal alignment of sequences for comparison is performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, or by Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970. By the homology alignment algorithms of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988 by the similarity search method, or by computer processing of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis. GAP, BESTFIT, FASTA and TFASTA) or by manual alignment and visual inspection (see, eg, Current Protocols in Molecular Biology (Ausubel et al., Eds 1995 supplement)). Can be done by.

Nucleotide: The basic unit of a nucleic acid molecule. Nucleotides contain nitrogen-containing bases attached to pentose monosaccharides with one, two or three phosphate groups attached to the sugar moiety by ester bonding.
The main nucleotides of DNA are deoxyadenosine 5'triphosphate (dATP or A), deoxyguanosine 5'triphosphate (dGTP or G), deoxycytidine 5'triphosphate (dCTP or C) and deoxythymidine 5'3. It is phosphoric acid (dTTP or T). The main nucleotides of RNA are adenosine 5'triphosphate (ATP or A), guanosine 5'triphosphate (GTP or G), cytidine 5'triphosphate (CTP or C) and uridine 5'triphosphate (UTP). Or U).
Nucleotides include such nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, as described, for example, in US Pat. No. 6,866,336.

Examples of modified base moieties that can be used to modify the nucleotide at any position in its structure include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5 -Iodouracil, hypoxanthin, xanthin, acetyl-citosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylue Osin, Inosin, N-6-Sopentenyl Uracil, 1-Methylguanin, 1-Methylinosin, 2,2-Dimethylguanine, 2-Methyladenine, 2-Methylguanin, 3-Methylcitosin, 5-Methylcitosine, N6 -Adenin, 7-Methylguanin, 5-Methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, β-D-mannosyleocin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-meth-ylthio- N6-isopentenyladenine, uracil-5-oxyacil, pseudouracil, queosin, 2-thiocitosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxy-methyl acetate Examples include esters, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine 2'-deoxyguanosine.
Examples of modified sugar moieties that can be used to modify nucleotides at any position in its structure include, but are not limited to, modified arabinose, 2-fluoroarabinose, xylose and hexose or phosphate skeletons. Includes components such as phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidat, phosphordiamidate, methylphosphonate or alkylphosphotriesters or similar thereofs.

Neuropeptides: Peptides released by neurons in the mammalian brain that specifically bind to neuropeptide receptors. Examples of neuropeptides include, among other things, melanin cell stimulating hormone (a-MSH), galanin-like peptide, cocaine-amphetamine regulated transcript (CART), neuropeptide Y, agouti-related protein (AGRP), (3-endolphin, dyno). Examples include neuropeptides, vasoactive intestinal peptides (VIPs) and substance P, including rufin, enkephalin, galanin, grelin, growth hormone-releasing hormone, neurotensin, neuromedin U, somatostatin, galanin, enkephalin cholesistokinin. The peptide or portion thereof is part of the disclosed functionalized electrode. In some embodiments, the antibody that specifically binds to the neuropeptide or portion thereof is part of the functionalized electrode, and thus in some examples. , The peptide can be used to detect the peptide in a sample.
Oligonucleotide: A linear polynucleotide sequence up to approximately 100 nucleotides in base length.

Parasites: Organisms that live within humans and other organisms that act as hosts (for parasites). Parasites depend on their host for at least part of their life cycle. Parasites are harmful to humans because they consume the necessary food, erode the tissues and cells of the body, and excrete toxic waste that makes people sick. Examples of parasites used according to the disclosure method and composition are, but are not limited to, Plasmodium falciparum, P vivax, P malariae. )), Schistosomes, Trypanosomes, Leishmania, Plasmodium falciparum, Trichomoniasis, Sarcosporidiasis, T. saginata ), T. solium), Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis (Eimedia species) Any one or more (or a combination thereof) may be mentioned. Thus, in some embodiments, the disclosed functionalized electrode comprises one or more antigens from one or more of the above organisms. In some embodiments, an antibody that specifically binds to one or more of the above organism-derived antigens is part of the disclosed functionalized electrode. Thus, in some examples, the disclosed functionalized electrodes can be used to detect the parasite in a sample, for example, to diagnose a particular parasite infection, or to detect the presence of a parasite in an environmental sample. can do.

Photoinitiator: An organic molecule or group that is cleaved into separate radical groups when irradiated with UV light. The molecule or group derived from α-hydroxy-, α-alkoxy- or α-amino-arylketone showing a 1-benzoyl-1-methyl-ethanol moiety is preferred; the most preferred photoinitiator is 2-hydroxy-. 4'-(2-Hydroxyethoxy) -2-methylpropiophenone and 2,2-dimethoxy-2-phenylphenone. "In the presence of photoinitiator" means that the photoinitiator is added in the form of a solution prior to the photoreaction or is previously attached to the metal surface, for example as disclosed by US Pat. No. 6,580,571. Means that.
Polypeptide: A polymer in which amino acid residues, which are monomers, are bound via an amide bond. When the amino acid is an a-amino acid, either the L-optical isomer or the D-optical isomer can be used. As used herein, the term "polypeptide" or "protein" is intended to include any amino acid sequence and to include modified sequences such as glycoproteins. "Polypeptides" include proteins of natural origin as well as proteins produced by recombination or synthesis. A "residue" or "amino acid residue" also includes a protein, polypeptide, or amino acid incorporated into the peptide.

Purified: The term "purified" does not require absolute purity; rather, it is meant to be a relative term. Thus, for example, purified peptides, proteins, conjugates, or other compounds are those that are wholly or partially isolated from the protein or other components of the mixture. Generally, substantially purified peptides, proteins, conjugates, or other active compounds used within the scope of the present disclosure are pharmaceutical carriers, excipients, etc. of peptides, proteins, conjugates or other active compounds. Consists of over 80% of all macromolecular species present in pre-formulation preparations or when mixed with buffers, absorption enhancers, stabilizers, preservatives, adjuvants or other co-ingredients. More typically, more than 90% of all macromolecular species present in the purified preparation before the peptide, protein, conjugate or other active compound has been purified and mixed with other pharmaceutical ingredients. In most cases, it is over 95%. In other cases, the purified preparation is inherently homogeneous and no other macromolecular species can be detected by conventional techniques.

Quantification: Determining or measuring the amount of a molecule (eg, relative quantity) or the activity of a molecule, eg, the amount of an analyte, eg, a target analyte present in a sample.
Sample: Substance to be analyzed. In one embodiment, the sample is a biological sample. In another embodiment, the sample is an environmental sample, such as soil, sediment, or air. Environmental samples are obtained from industrial sources such as farms, waste logistics, or water sources. A biological sample is a sample containing a biological substance (eg, nucleic acid and protein). In some examples, the biological sample is obtained from an organism or a portion thereof, such as an animal. In certain embodiments, the biological sample is obtained from an animal subject, such as a human subject. The biological sample is, but is not limited to, any solid or fluid sample obtained from any living organism, including, but not limited to, multicellular organisms (eg, animals), or excreted or secreted by any living organism. (Including samples from healthy or apparently healthy human subjects or human patients with conditions or diseases such as cancer to be diagnosed or investigated). For example, biological samples are biology obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, atrioventricular or vitreous fluid, or any body secretions, leaks, exudates. Fluids (eg, fluids obtained from abscesses or any other site of infection or inflammation), or joints (eg, normal joints or joints suffering from diseases such as rheumatoid arthritis, osteoarthritis, gout or purulent arthritis). It may be a fluid obtained from. The biological sample may be a sample (including a biopsy or autopsy sample, eg, a tumor biopsy) obtained from any organ or tissue, or a cell (primary cell or cultured cell) or either. It may also include a medium conditioned by cells, tissues or organs. In some examples, the biological sample is a cytolytic product, eg, a cytolytic product obtained from a tumor of interest.

Spacer group: A group in the cross-linker that separates the two terminal reactive groups. Upon irradiation, one of the reactive groups binds to the metal surface and the other to the sulfhydryl group of the biomolecule. The spacer group is preferably a C _5-30 alkylene-di-1, ω-yl group, with one or more non _- adjacent CH groups independently O, S, NH, NR, NR-CO, CO- It may be replaced with a group selected from NR, O-CO and CO-O (R stands for hydrogen or C _1-6 alkyl). A polyethylene glycol (PEG) group is a preferred component of the spacer group.
Specific binding factor: A factor that binds substantially only to a defined target. Therefore, an antigen-binding factor such as an antibody that is specific to an antigen is a factor that substantially binds to a specific antigen or a fragment thereof. In some examples, the specific binding factor is a monoclonal or polyclonal antibody that specifically binds to a specific antigen or an antigenic fragment thereof, such as a target analyte. In another example, the specific binding factor is an antigen that specifically binds to an antibody specific for the antigen. In some examples, the specific binding factor is bound to an enzyme, eg, an enzyme that catalyzes the reaction of an enzyme substrate into an electrically active product.
Subjects: Both human and veterinary subjects, such as humans, non-human primates, dogs, cats, horses, pigs, and cattle, as well as subjects in the food production sector, such as chickens or fish, such as salmon, tuna, or trout. included. Another important target in the food production sector is plant material.

Substrate: A molecule that is acted upon by an enzyme. The substrate binds to the active site of the enzyme to form an enzyme-substrate complex. In some examples, the enzymatic substrate is enzymatically converted to an electrically active product.
Thiol: An organic sulfur compound containing a sulfur-hydrogen bond or a sulfhydryl group (SH). Thiol is a sulfur analog of alcohol. The SH functional group may be referred to as a thiol group or a sulfhydryl group. Thiols have the general chemical formula RSH. In some examples, SH groups can be attached to a surface, such as a conductive surface, by reacting with the surface.

Tumor antigens: Tumor antigens are antigens produced by tumor cells that can stimulate a tumor-specific T-cell immune response. Exemplified tumor antigens include, but are not limited to, RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1, melan-A / MART-1, glycoprotein (gp) 75, gp100, Included are β-catenin, melanoma preferential expression antigen (PRAME), MUM-1, Wilms tumor (WT) -1, carcinoembryonic antigen (CEA), and PR-1. Further tumor antigens are known in the art (see, eg, Novellino et al., Cancer Immunol. Immunolother. 54 (3): 187-207, 2005), which will be discussed later. Tumor antigens are also referred to as "cancer antigens". The tumor antigen may be any of the technically well known tumor-related antigens, such as carcinoembryonic antigen (CEA), (3-human villous gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE. -1, MN-CA IX, human telomerase reverse transcript, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, macrophage colony stimulator, prostase, prostate-specific antigen (PSA), PAP, NY -ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1, MAGE, ELF2M, neutrophil elastase, efrin B2, CD22, insulin-like Growth factors (IGF) -I, IGF-II, IGF-I receptors and mesothelin are included.
In some embodiments, the tumor antigen or portion thereof is part of the disclosed functionalized electrode. In some embodiments, an antibody that specifically binds to the tumor antigen or portion thereof is part of the disclosed functionalized electrode. Thus, in some examples, the disclosed functionalized electrodes can be used to detect the antigen in a sample and, for example, to diagnose cancer.

Virus: A microscopic-level infectious organism that grows in living cells. The virus consists essentially of a nucleic acid core surrounded by a protein coat and has the ability to replicate only within living cells. "Viral replication" is the production of additional viruses by the presence of at least one viral life cycle. The virus can exterminate the normal functioning of the host cell and allow the cell to behave in a manner determined by the virus. For example, viral infection can give rise to cells that produce or respond to cytokines, whereas non-infected cells normally do not produce cytokines. In some cases, the virus is a pathogen. Another form of virus is a prion.

Specific examples of viral pathologies used according to the disclosure method and composition are, but are not limited to, arenaviruses (eg, guanalitovirus, lassavirus, funinevirus, machupovirus and saviavirus), arterivirus, loni. Virus (Ronivirus), Astrovirus, Bunyavirus (eg Crimea Congo hemorrhagic fever virus and Hunter virus), Barnavirus, Burnavirus, Bornavirus (eg Borna disease virus), Bromovirus, Calisivirus, Chrysovirus (eg) Chrysovirus), coronavirus (eg coronavirus and SARS), cystovirus, crossterovirus, comovirus, Dicistroviruses, flavirus (eg yellow fever virus, Westnile virus, hepatitis C virus, etc.) And dengue virus), phyllovirus (eg Ebola virus and Marburg virus), flexivirus, hepevirus (eg hepatitis E virus), human adenovirus (eg human adenovirus AF), human astrovirus, human BK polyoma Virus, human bocavirus, human coronavirus (eg human coronavirus HKU1, NL63, and 0C43), human enterovirus (eg human enterovirus AD), human erythrovirus V9, human formy virus, human herpesvirus (eg human herpesvirus 1 (eg human herpesvirus 1) Simple herpesvirus type 1), human herpesvirus 2 (simple herpesvirus type 2), human herpesvirus 3 (hydropultic herpes zoster virus), human herpesvirus type 4 1 (human herpesvirus 4 type 1) (Epstein bar virus type 1) (Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barr virus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5 strain Merlin strain, human Herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus 7, Human herpesvirus 8 type M, Human herpesvirus 8 type P and human cytomegalovirus), Human immunodeficiency virus ( HIV) (eg HIV 1 and HIV 2), human metapneumovirus, human papillomavirus, human parainfluenza virus (eg human parainfluenza virus 1-3), human parechovirus, human parvovirus (eg human parvo) Virus 4 and human parvovirus B19), human respiratory polynuclear virus, hitrinovirus (eg, hitrinovirus A and hitrinovirus B), human spumaretrovirus, human T lymphophilic virus (eg,) Human T lymphophilic virus 1 and human T lymphophilic virus 2), human polyomavirus, hypovirus, levivirus, luteovirus, lymphocytic choriomyelitis virus (LCM), Marnavirus ), Narnavirus, Nidovirales, Nodavirus, Orthomixovirus (eg influenza virus), Partitivirus, Paramixovirus (eg measles virus and epidemic parotitis virus) , Picornavirus (eg, poliovirus, cold virus, and hepatitis A virus), potivirus, poxvirus (eg, acne and bovine hemorrhoids), sekivirus, leovirus (eg, rotavirus), rabdovirus (eg, mad dog disease virus) , Rabdovirus (eg, bullous stomatitis virus), Tetravirus, Togavirus (eg, ruin virus and Ross River virus), Tombus virus, Totivirus, Timovirus, and norovirus. Combination).

The viral antigen can be derived from hepatitis C virus (HCV). The HCV antigen can be selected from one or more of El, E2, El / E2, NS345 polyproteins, NS345 core polyproteins, cores and / or peptides from non-structural regions (Houghton et al. (1991 incorporated by reference). ) Hepatology 14: 381-388).
The viral antigen can be derived from human herpesvirus, such as herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-bar virus (EBV), or cytomegalovirus (CMV). The human herpesvirus antigen can be selected from the earliest protein, the early protein, and the late protein. The HSV antigen may be derived from the HSV-I or HSV-2 strain. The HSV antigen can be selected from the glycoproteins gB, gC, gD and gH, or the immune escape protein (gC, gE, or gl). The VZV antigen can be selected from core, nucleocapsid, segmentation, or enveloped proteins. Live attenuated VZV vaccines are commercially available. The EBV antigen can be selected from the glycoproteins of the initial antigen (EA) protein, the viral capsid antigen (VCA), and the membrane antigen (MA). The CMV antigen can be selected from capsid proteins, enveloped glycoproteins (eg gB and gH), and segmentation proteins. Illustrative herpes antigens include human herpesvirus 1 (herpes simplex virus type 1) (NC 001806), human herpesvirus 2 (herpes simplex virus type 2) (NC 001798), and human herpesvirus 3 (herpes zoster virus) (NC). 001348), human herpesvirus type 4 1 (Epstein-barvirus type 1) (NC 007605), human herpesvirus type 4 2 (Epstein-barvirus type 2) (NC 009334), human herpesvirus 5 strain AD169 (NC 001347) ), Human herpesvirus 5 strain Merlin strain (NC 006273), human herpesvirus 6A (NC 001664), human herpesvirus 6B (NC 000898), human herpesvirus 7 (NC 001716), human herpesvirus type 8 M (NC 003409) and those derived from human herpesvirus type 8 P (NC 009333) (GENBANK ™, acceptance number in parentheses).

Human papillomavirus (HPV) antigens are technically well known, eg, International Patent Publication No. W096 / 19496, which discloses variants of the E6 / E7 fusion protein with deletions in both the HPV E6 and E7 proteins, especially the E6 and E7 proteins. Found by issue (the whole is incorporated by reference). Antigens based on HPV L1 are all disclosed in International Patent Publications W094 / 00152, W094 / 20137, W093 / 02184 and W094 / 05792, which are incorporated by reference. The antigen may include the Ll antigen as a monomer, capsomere or virus-like particle. The particles may further contain L2 protein. Other HPV antigens are early proteins such as E7 or fusion proteins such as L2-E7. Exemplified HPV antigens include human papillomavirus-1 (NC 001356), human papillomavirus-18 (NC 001357), human papillomavirus-2 (NC 001352), human papillomavirus-54 (NC 001676), human papillomavirus-61 (NC 001694). , Human papillomavirus-cand90 (NC 004104), human papillomavirus RTRX7 (NC 004761), human papillomavirus type 10 (NC 001576), human papillomavirus type 101 (NC 008189), human papillomavirus type 103 (NC 008188), human papillomavirus type 107 (NC 008188) NC 009239), human papillomavirus type 16 (NC 001526), human papillomavirus type 24 (NC 001683), human papillomavirus type 26 (NC 001583), human papillomavirus type 32 (NC 001586), human papillomavirus type 34 (NC 001587), human papillomavirus Virus type 4 (NC 001457), human papillomavirus type 41 (NC 001354), human papillomavirus type 48 (NC 001690), human papillomavirus type 49 (NC 001591), human papillomavirus type 5 (NC 001531), human papillomavirus type 50 (NC) 001691), human papillomavirus type 53 (NC 001593), human papillomavirus type 60 (NC 001693), human papillomavirus type 63 (NC 001458), human papillomavirus type 6b (NC 001355), human papillomavirus type 7 (NC 001595), human papillomavirus Type 71 (NC 002644), human papillomavirus type 9 (NC 001596), human papillomavirus type 92 (NC 004500), and human papillomavirus type 96 (NC 005134) (GENBANK ™, acceptance number in parentheses) Can be mentioned.

The viral antigen may be derived from a retrovirus such as oncovirus, lentivirus or spumavirus. The oncovirus antigen can be derived from HTLV-I, HTLV-2 or HTLV-5. The lentiviral antigen can be from HIV-1 or HIV-2. Retroviral antigens can be selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. Antigens for HIV are technically well known, for example HIV antigens are selected from gag (p24gag and p55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins, (p55 gag and gp140v). Get it. The HIV antigen may be derived from one or more of the following strains: HIVmb, HIV; HIVVAV, HIVLAI, HIVM N, HIV-1 CM235, HIV-1 US4. Examples of HIV antigens can be found in International Patent Publications WO 09 / 089,568, WO 09 / 080,719, WO 08 / 099,284, and WO 00/15255, as well as US Pat. Nos. 7,531,181 and 6,225,443. Will be. All of these are incorporated by reference. Examples of HIV antigens include those derived from human immunodeficiency virus 1 (NC 001802) and human immunodeficiency virus 2 (NC 001722) (GEN-BANK ™, acceptance number in parentheses).

In some embodiments, the disclosed functionalized electrode comprises one or more antigens from one or more of the above viruses. In some embodiments, an antibody that specifically binds to one or more virus-derived antigens described above is part of the functionalized electrode. Thus, in some examples, the disclosed functionalized electrodes can be used to detect the virus in the sample, eg, to diagnose a viral infection or to detect the presence of the virus in an environmental sample.
The general performance of electrochemical sensors is often determined by the surface architecture that connects the sensing element to the biological sample on the nanometer scale. Electrochemical sensors suffer from a lack of surface architecture that allows sufficiently sensitive and unique identification of responses to desired biochemical events.

Various previous attempts have been made from the self-assembled monolayer (SAM) due to the desirable properties of the self-assembled monolayer, such as stability and resistance to non-specific biomolecular adsorption. I've been trying to create a biosensor. However, long-chain alkyl-based electrochemical sensors have suffered from limited applicability due to their low permeability to electron transfer (eg Fragoso et al., Anal. Chem., 80: 2556-). See 2563, 2008). In an attempt to overcome the perceived limitations of long-chain SAMs, Fragoso et al. Focused on dithiol, which is considered to be poorly insulated. However, by switching to a low-insulation monomolecular layer, one advantage of using a long-chain SAM is lost. That is, the loss of selectivity for non-specific electron transfer reduces the signal for sensor noise and thus the sensitivity.
Another drawback to the use of SAMs is that they are ionic insulators, which make it easy for ions to transfer electrons to and from the conductive material underlying the electrochemical sensor. It is impervious (see, for example, Boubour and Lennox, Langmuir 16: 42224228, 2000). Insulation properties of SAM are desirable from the standpoint of limiting non-specific electron transfer, but in the absence of selective ion transfer for the analyte of interest, SAM is limited in its use as a component of electrochemical sensors. Will be done.

As disclosed herein, their insulating properties towards non-specific electron transfer, coupled with the selection of enzymatic reaction products that are electrically active and capable of facilitating electron transfer through the monolayer to the electron conduction surface. Careful selection of retained thiol or dithiol compounds overcomes the limitations of previous attempts to create sensors from long chain thiol-containing SAMs. Thus, as disclosed herein, it has been surprisingly found that functionalized electrodes can be formed that retain SAM-based beneficial insulation properties, such as resulting in high signal-to-noise, with high selectivity and sensitivity. ..
Kits are also provided herein. The kit for detecting the analyte of interest contains one or more disclosed biosensors. In some embodiments, the kit contains instructions that disclose the means of detecting the analyte of interest. The instructions may be written in electronic form (eg computer diskettes or compact discs) or may be visual (eg video files). The kit also contains a detection reagent and substrate with an electrically active reaction product. The kit may also contain additional ingredients to facilitate the particular application for which the kit is designed. Thus, for example, the kit may further contain buffers and other reagents that are routinely used to carry out certain methods. The kit and suitable contents are technically well known. In some examples, the kit comprises a control solution containing, for example, a control, eg, a known amount or concentration of target analyte, as a means of calibrating the biosensor included in the kit. The kit may include components for automated assay testing and automated data acquisition. These will be useful for a quick point-of-care setting.

A preferred embodiment of the method of the invention is
The (A) cross-linker compound is a compound of the following formula (I).
HS- (CH ₂ ) _m -CH (ZH) -SPACER- (CH ₂ ) _p -A (I)
(During the ceremony,
m is an integer from 2 to 6, especially an integer from 2 to 4.
A is selected from -CH = CH ₂ and -C≡CH, especially -CH = CH ₂ , and
Z is S or a single bond, especially S,
SPACER is the following formula
-(CH ₂ ) _n- (C = O) _x -Y-(CH ₂ CH ₂ -O) _y- (CH ₂ ) _r- (C = O) _v -X-
Is the basis of
here,
X and Y are independently NH or O, respectively.
n is an integer of 0 or 1-10, particularly an integer of 2-6, most preferably 4.
x and v are independently 0 or 1, especially 1 and
y is an integer from 1 to 20, especially an integer from 4 to 18.
r and p are each independently selected from integers 1-6, especially 1;
The polyethylene glycol group (PEG)-(CH ₂ CH ₂ -O) _y -contains a specified number of ethylene oxide units, particularly 4-18 units, most preferably 8-16 units, or 0.5-10 kDa, in particular. Contains an uncertain number of ethylene oxide units with an average molecular weight of 2-8 kDa, most preferably about 5 kDa);
(B) Biomolecules are antibodies, enzymes or nucleic acids, especially monoclonal antibodies;
(C) The photoinitiator is a 1-benzoyl-1-methyl-ethanol derivative, particularly a water-soluble 1-benzoyl-1-methyl-ethanol derivative, most preferably 2-hydroxy-4- (2-hydroxyethoxy) -2. -Methylpropiophenone;
(D) Irradiation is performed at a wavelength of 300 to 340 nm, especially about 320 nm at a wavelength of λ _max .

Further, in the present invention, the novel compound of the formula (I), preferably the compound of the formula (I) is the following formula (IA) :.
HS- (CH ₂ ) _m -CH (SH) -SPACER-CH ₂ -A (IA)
(In the equation, A and SPACER have the meaning given for equation (I) and
m is an integer from 2 to 4, especially 2.
The SPACER of equation (IA) is preferably the following equation:
-(CH ₂ ) _n- (C = O) -NH-(CH ₂ CH ₂ -O) _y -CH _2- (C = O) -NH-
Is the basis of
here,
n is an integer of 0 or 1-10, particularly an integer of 2-6, most preferably 4.
y is an integer from 1 to 20)
It is a compound of.

Most preferably, from liponic acid, especially from R-α-lipoic acid, the following formula (IB): obtained by reduction of -SS- bonds:
HS- (CH ₂ ) ₂ -CH (SH)-(CH ₂ ) _4- (C = O) -NH-(CH ₂ CH ₂ -O) _y -CH _2- (C = O) -NH-CH ₂ -A (IB)
(In the formula, A and y have the meaning given for formula (I))
It is a compound of.
The preferred compounds of formulas (IA) and (B) can be synthesized according to the following reaction scheme I.
Reaction scheme I

In step 1) and step 2), the corresponding acid is treated with an alcohol (Y or X = O) or an amine (Y or X = NH) under the conditions of an esterification reaction or an amide formation reaction. The reaction conditions are well known to those of skill in the art. The acid is activated, for example by treatment with N-hydroxysuccinimide (NHS) or thionyl chloride, and / or the water produced during the reaction is irreversibly trapped by a dehydrating agent.
Most preferably N-hydroxysuccinimide (NHS), sodium N-hydroxysulfosuccinimide, 1-hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT) or pentafluorphenol and dicyclohexylcarbodiimide (DCC). Alternatively, the acid is treated with an alcohol or amide in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).
In step 3), treatment with a reducing agent, especially DTT (dithiothreitol), mercaptoethanol, tris (2-carboxyethyl) phosphine (TCEP) and / or DTE (dithioerythreitol) to reductively cleave the disulfide bond. ..
The preferred compound of formula I, where X is S and v is 0, can be prepared according to the following reaction scheme II.
Reaction scheme II

The protecting group PG is a group suitable for protecting an amino or hydroxyl group. The protecting group and the deprotection method (step 2) are well known to those skilled in the art. Compare with TW Green, PGM Wuts, Protective Groups in Organic Synthesis, Wiley-Interscience, New York, 1999. The most preferred protecting groups for amino groups are, for example, 9-fluorenyl-methylcarbamate, (FMOCamino), t-butylcarbamate (BOCamino), benzylcarbamate, acetamide, trifluoroacetamide, benzylamine and tritylamine. be. The most preferred protecting group for hydroxy is, for example, methoxymethyl ether, tetrahydropyranyl ether, t-butyl ether, benzyl ether, trihydrocarbylsilyl ether, such as t-butyldimethylsilyl ether or t-butyldiphenylsilyl ether.
In principle, the substitution reaction in step 3) is carried out using an alkali metal hydroxide, preferably sodium hydroxide or potassium hydroxide, or a tertiary amine, preferably a base such as triethylamine or 2,2,6,6-tetramethylpiperidine. Do in the presence.
Step 4 can be carried out similar to the description of reaction scheme 1 and step 3).
Therefore, novel lipoic acid derivatives of formula (II) are another subject of the invention as intermediates for the preparation of compounds of formulas (I), (IA) and (IB).
Compound of formula (IIA) below

In particular, the compound of the following formula (IIA1)

Is the most preferable
In the equation, SPACER, A and p have the meaning given for equation (I).

Examples The present disclosure will be described with reference to the following non-limiting examples.
Abbreviation
AcOH acetic acid
approx. Approx. ℃ degrees Celsius
DCC dicyclohexylcarbodiimide
DCM Dichloromethane
DTT Dithiothreitol
kDa kilodalton
MeCN acetonitrile
MeOH Methanol
mL ml
mM mmol concentration
NHS N-hydroxysuccinimide
PEG-Alkene methoxypoly (ethylene glycol) alkene
PEG-alkyne methoxypoly (ethylene glycol) alkyne
rpm rpm λ wavelength
TCEP Tris (2-carboxyethyl) phosphine
TEA Triethylamine
TFA trifluoroacetic acid

Synthesis of SH-PEG-alkene / -alkyne crosslinker PEG-alkene and PEG- with average molecular weights of 800 Da and 5000 Da for coupling of diagnostic biomolecules to microchips by PEG-alkene / -alkyne crosslinker An alkyne molecule was synthesized. In addition, 600 Da SH-mPEG that was not functionalized with an alkene or alkyne was synthesized. This molecule can be used to dilute PEG-alkene / -alkyne crosslinkers in metal surface coatings to obtain a uniform statistical distribution of functional groups on the chip surface.

experiment
RP-HPLC-RP-HPLC analysis was performed on a Water Alliance system equipped with an ELSD detector and a photodiode array detector. An XBridge ™ BEH300 4.6 × 250 mm 5 μm RP18 column was used. HPLC-H ₂ O (+ 0.1% TFA) and HPLC-MeC N (+ 0.1% TFA) were used as eluents for gradient HPLC.
For NMR-NMR spectroscopy, 30-60 mg of the analyte was dissolved in 600 μL CDCl3 and transferred to an NMR vial. Spectroscopy was performed using Varian Mercury-400BB at (1H, 400MHz; 13C, 100.6MHz) or Varian Mercury-300BB at (1H, 300MHz; 13C, 75.5MHz). Chemical shifts were expressed in ppm and calibrated to solvent signals.
MALDI-TOF-MS analysis was performed in linear mode using the α-cyano-4-hydroxy-silicic acid (CHCA) matrix on the MALDI-TOF-MS-Axima Confidence System (Shimadzu).
Stirrer-Magnetic stirrer bars were added to all reaction mixtures and crystallization suspensions and they were stirred with MR3001 K (Heidolph) magnetic stirrer.
Evaporation and drying-Laborota 4000 with Vacuum Pump Unit PC510 (Vacuu brand) and a water bath at a temperature of 40 ° C.-The solvent was removed and dried under reduced pressure using an efficient (Heidolph) rotary evaporator. The same procedure was used to dry the material.
Chromatography-Chromatography was performed with a mixture of different typical organic solvents using 40-63 μm silica gel packed in a glass column. Fractions were manually collected and analyzed by TLC. The pure product-containing fractions were mixed and the solvent removed under reduced pressure.
TLC analysis was performed on a TLC-TLC silica gel 60 F254 aluminum sheet using different mixtures of typical organic solvents. An aqueous solution of potassium permanganate was used as a staining reagent.

Example 1.1.
Synthesis of R-α-lipoic acid-PEG12-allyl (B)

Step 1: Add DCC (1.00 g, 4.86 mmol) and NHS (599 mg, 4.86 mmol) to a solution of R-α-lipoic acid (668 mg, 3.24 mmol) in 40 mL DCM and allow the resulting mixture to cool at room temperature for 1 hour. Stirred. The precipitated dicyclohexylurea was removed by filtration and washed twice with 10 mL of DCM. NH ₂ -PEG12-COOH (2.00 g, 3.24 mmol) and TEA (900 μL, 6.48 mmol) were added to the obtained clear yellow reaction mixture. The reaction mixture was stirred at room temperature for 2.5 hours. The solvent was subsequently removed under reduced pressure. 1.99 g product showing 76% yield after purification by chromatography of the resulting residue (35 g silica gel (40-63 μm) DCM / MeOH-98: 2 → 90:10, + 0.1% AcOH) Obtained (A). According to RP-HPLC analysis, the product contained approximately 3.3% lipoic acid and up to 2.8% unspecified impurities. The overall purity was about 94%.

Step 2: DCC (461 mg, 2.23 mmol) and NHS (257 mg, 2.23 mmol) were added to a solution of the product (A) (1.20 g, 1.49 mmol) obtained in Step 1 in 20 mL of DCM, and the obtained reaction was obtained. The mixture was stirred at room temperature for 1 hour. The precipitated dicyclohexylurea was removed by filtration and washed twice with 5 mL of DCM. Allylamine (224 μL, 2.98 mmol) and TEA (415 μL, 2.99 mmol) were added to the obtained clear yellow reaction mixture. The reaction mixture was stirred at room temperature for 17 hours and subsequently washed with 20 mL of water. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (15 g silica gel (40-63 μm) DCM / MeOH-98: 2 → 95: 5). 796 mg of product CES0594 with a yield of 63% was obtained. According to RP-HPLC analysis (Fig. 2), the product had a purity of> 99%. The molecular weight and identity of (B) were confirmed by MALDI-MS and NMR.

Example 1.2.
Synthesis of R-α-lipoic acid-PEG12-propargyl (C)

DCC (592 mg, 2.87 mmol) and NHS (325 mg, 2.82 mmol) were added to a solution in 40 mL of DCM of the product (A) (1.52 g, 1.89 mmol) obtained in step 1 of Example 1.1. The resulting reaction mixture was stirred at room temperature for 1 hour. The precipitated dicyclohexylurea was removed by filtration and washed twice with 5 mL of DCM.
Propargylamine (245 μL, 3.82 mmol) and TEA (525 μL, 3.79 mmol) were added to the obtained clear yellow reaction mixture. The reaction mixture was stirred at room temperature for 1 hour and subsequently washed with 20 mL of water. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (21 g silica gel (40-63 μm) DCM / MeOH-98: 2 → 95: 5). 1.01 g of product CES0596 with a yield of 64% was obtained. According to RP-HPLC analysis, the product did not contain detectable impurities. The molecular weight and identity of (C) were confirmed by MALDI-MS and NMR.

Example 1.3.
Synthesis of R-α-lipoic acid-mPEG8 (D)

DCC (8.11 mg, 3.93 mmol) and NHS (450 mg, 3.91 mmol) were added to a solution of R-α-lipoic acid (538 mg, 2.61 mmol) in 40 mL of DCM, and the resulting reaction mixture was stirred at room temperature for 1 hour. did. The precipitated dicyclohexylurea was removed by filtration and washed twice with 5 mL of DCM. NH2-mPEG8 (0.98 g, 2.56 mmol) and TEA (725 μL, 5.23 mmol) were added to the obtained clear yellow reaction mixture. The reaction mixture was stirred at room temperature for 1 hour and subsequently washed with 30 mL of water. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (34 g silica gel (40-63 μm) DCM / MeOH-98: 2 → 97: 3). 1.12 g of product (D) with a yield of 75% was obtained. According to RP-HPLC analysis, the product had a purity of about 99.8%. The molecular weight and identity of CES0598 were confirmed by MALDI-MS and NMR.

Example 1.4.
Synthesis of R-α-lipoic acid-5kDa PEG-allyl (F)

Step 1: DCC (184 mg, 892 μmol) and NHS (108 mg, 938 μmol) were added to a solution of R-α-lipoic acid (125 mg, 606 μmol) in 5 mL of DCM, and the obtained reaction mixture was stirred at room temperature for 1 hour. The precipitated dicyclohexylurea was removed by filtration and washed twice with 2 mL of DCM. NH2-5kDa PEG-COOH (3.03 g, 606 μmol) and TEA (166 μL, 1198 μL) in DCM (91 mL) were added to the obtained clear yellow reaction mixture. The reaction mixture was stirred at room temperature for 17 hours and subsequently washed with 50 mL of 100 mM citric acid. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (36 g silica gel (40-63 μm) DCM / MeOH −98: 2 → 90: 10 + 0.1% AcOH). 2.75 g of product (E) with 88% yield was obtained. According to RP-HPLC analysis, the product had about 80% purity and 20% unknown impurities. These impurities were removed during the purification of the next step.

Step 2: DCC (76 mg, 368 μmol) and NHS (42 mg, 365 μmol) were added to a solution of (E) (1.30 g, 251 μmol) in 40 mL of DCM, and the obtained reaction mixture was stirred at room temperature for 6 hours. Allylamine (50 μL, 666 μmol) and TEA (100 μL, 721 μmol) were added to the slightly cloudy yellow solution and the mixture was stirred at room temperature for 18 hours. Precipitated dicyclohexylurea and free N-hydroxysuccinimide were removed by filtration and washed twice with 10 mL DCM. The reaction mixture was washed with 30 mL of water. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (31 g silica gel (40-63 μm) DCM / MeOH-95: 5 → 80:20). 1.18 g of product (F) showing 90% yield was obtained. According to RP-HPLC analysis, the product had a purity of about 97.5%. The molecular weight and identity of (F) were confirmed by MALDI-MS and NMR.

Example 1.5.
Synthesis of R-α-lipoic acid-5kDa PEG-propargyl (G)

DCC (116 mg, 562 μmol) and NHS (59 mg, 513 mmol) were added to a solution in 40 mL of DCM of the product (E) (1.30 g, 251 μmol) obtained according to Step 1 of Example 1.4, and the obtained reaction mixture was added. The mixture was stirred at room temperature for 3.5 hours. Propargylamine (48 μL, 749 μmol) and TEA (104 μL, 750 μmol) were added to the yellow slightly cloudy solution and the mixture was stirred at room temperature for 20 hours. Precipitated dicyclohexylurea and free N-hydroxysuccinimide were removed by filtration and washed twice with 10 mL DCM. The reaction mixture was washed with 30 mL of water. The organic phase was concentrated under reduced pressure and the resulting solid was purified by chromatography (32 g silica gel (40-63 μm) DCM / MeOH-95: 5 → 80:20). 1.18 g of product (G) with 90% yield was obtained. According to RP-HPLC analysis, the product had a purity of about 95%. The molecular weight and identity of (G) were confirmed by MALDI-MS and NMR.

Example 2.1. Investigation of Photochemical Coupling Conditions A photochemical reaction between R-α-lipoic acid-PEG12-allyl (B) and 3-mercaptopropionic acid was established using a model system (Scheme 7):

The photochemical modification of the partially reduced antibody was investigated using the optimization conditions.
Photoinitiator II (2,2-dimethoxy-2-phenylphenone) showed inadequate solubility in aqueous solution (<1 mg / 1 mL 10% DMSO aqueous solution), so photoinitiator I (2) All experiments were performed using only -hydroxy-4'-(2-hydroxyethoxy) -2-methylpropiophenone). In contrast, photoinitiator I was dissolved in water to a concentration of 1 mg / mL without the addition of DMSO.
In the initial experiment, a stock solution of product (B) at 1 mg / ml in water was treated with 10 mol% photoinitiator I. The reaction mixture was divided into 5 tubes (200 μl per tube, A to E). Different molar excesses of 3-mercaptopropionic acid (SH) were added to these tubes (see Table 1, entries A-E).

Table 1

The reaction was triggered using the following conditions.
・ Reaction temperature = 1 ℃
・ Light exposure time = 30 minutes ・ λ _max : 310 nm
Immediately after irradiation, the sample was analyzed by HPLC. In short, these experiments demonstrated the feasibility of photo-induced coupling of thiol reagents to the alkyne group of the synthesized cross-linker.
Further experiments were performed to determine the dependence on reaction rate and reaction volume. In 300 seconds, a conversion of about 60% was obtained and linear regression well described the reaction process. The conversion rate did not depend on the applied reaction volume. A reaction volume of 20 μL or 200 μL resulted in the conversion rate at the same reaction time. In contrast, the typical kinetics of the reaction type (Macromol. Rapid Commun. 2010, 31, 1247-1266) show a logarithmic correlation. This feasibility study focused on the reproducibility of light-induced click responses rather than complete conversion. At this point, a reproducible conversion rate of 2% was already obtained after 5 seconds.
Furthermore, based on these results, a first experiment with a reducing antibody was designed. Since each reaction product can be detected by SDS-PAGE due to the mass difference, only 5 kDa PEG conjugates (alkene (F) and alkyne (G)) were used for reaction progress.

3.1. Partial reduction of antibody A free thiol group can be introduced into an antibody by partial reduction of the internal disulfide bond of the antibody. Therefore, reduction conditions that result in partial reduction of the antibody without affecting antibody activity should be investigated. The reaction should be carried out in a volume of 25-50 μL. DTT, TCEP and cysteamine should be tested as reducing agents. The partially reduced antibody should then be modified with a 5 kDa mPEG-maleimide that reacts with a free sulfhydryl group to investigate the success of the reduction reaction.

3.2. Experimental Reducing Agent Screening-Reducing agents at protein concentrations of 0.5 mg / mL (anti-ACTH antibody) or 0.5, 5, 10 and 15 mg / mL (IgG1) with various reducing agent excesses as shown in the Results section. A screening experiment was performed. The reaction was carried out at room temperature for 1 or 2 hours with a final volume of 25 μL using 20 mM sodium phosphate and pH 7.2 as a reaction buffer.
PEGylation of antibody-PEGylation reaction was carried out at room temperature for 3 hours at a PEG: protein molar ratio of 400: 1 in 20 mM Na phosphate, pH 7.2. Protein concentrations in the reaction mixture ranged from 0.3 mg / mL (anti-ACTH antibody) to 0.3, 3, 6 and 9 mg / mL (IgG1).
SDS-PAGE-under reducing or non-reducing conditions, SDS-PAGE was performed using a Bis-Tris gel of 4-12% of the NuPAGE system from Invitrogen. The gel was stained with the SimplyBlue ™ Safe Stain Kit (Invitrogen).
Size Exclusion Chromatography-SEC was performed on an Agilent 1200 with a Superdex 200 10/300. SEC was performed using PBS (10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , 137 mM NaCl, 2.7 mM KCl, pH 7.4) as an eluent at a flow rate of 0.65 mL / min.

3.2. Results In the initial experiment, partial reduction of anti-ACTH antibody was investigated using different concentrations of reducing agents. In this way, the concentration of reducing agent capable of obtaining a completely non-dissociated IgG should be determined.
Using common mouse IgG1 antibodies (Biogenes), conditions suitable for partial reduction were investigated in more detail.
In particular, the effect of IgG1 concentration on reduction efficiency was investigated for DTT. 5 kDa mPEG-maleimide was added to the reaction mixture in excess (400-fold) to quantitatively detect all resulting free thiol groups as bandshifts on SDS-PAGE.
In general, higher proportions of reduced IgG1 were detected as the excess amount of reducing agent increased. In addition, higher protein concentrations required lower DTT excesses to induce reduction of IgG1. Using 6 or 10 equivalents of DTT, SDS-PAGE analysis yielded 0-5 fold modification of the heavy chain with 5 kDa mPEG-maleimide. Reduction with 1.5 or 3 equivalents of DTT predominantly resulted in heavy chain monoPEGylation, but to a lesser extent, higher modifications were also observed. At a ratio of 1% (1.5 eq DTT) to about 20% (10 eq DTT), only monomodification of the light chain was observed.
Furthermore, the more the reduced form of IgG1, the higher the degree of PEGylation. The effect of disulfide reduction on the association state of IgG1 after the addition of the reducing agent and PEG was further analyzed by SEC.
Non-reduced IgG1 was eluted as a single and typical peak at 20.1 minutes. Treatment with a 10-fold excess of DTT had no detectable effect on antibody retention time or profile. This suggests completeness under physiological conditions. This finding was in contrast to the SDS-PAGE findings in which dissociation of IgG1 into heavy and light chains was observed under the same conditions.

Modification of partially reduced IgG1 (10-fold excess DTT) with 5 kDa mPEG-Mal extended the peak to shorter retention times of 14.5-22 minutes. This was predicted for a complete antibody modified with 5 kDa mPEG. No signal was observed with a larger retention time that would indicate antibody dissociation. At the lower DTT excess (1.5-fold), the peaks obtained during SEC were less widespread than the 10-fold DTT excess. This suggests a lower degree of PEGylation.
Using anti-ACTH antibody, the effect of reduction time was investigated for all three reducing agents with various molar excesses. After 1 and 2 hours of incubation, a high molar excess of 5 kDa mPEG-maleimide was added to stop the reduction.
Complete anti-ACTH antibody was observed by SDS-PAGE under all investigation conditions. This suggests an incomplete reduction of the interchain disulfide bond. However, as the excess amount of DTT or TCEP increased, higher proportions of heavy and light chains as well as higher degrees of PEGylation were detected. This effect was slightly stronger with a reduction time of 2 hours. With cysteamine, it was possible to achieve no significant dissociation of the anti-ACTH antibody with a 300-900-fold excess reducing agent. Based on these results, we decided to use a reduction time of 1 hour. In addition, TCEP was selected as the reducing agent for the preparation of PEGylated anti-ACTH antibodies because TCEP is not modified with maleimide-activating molecules.

Example 4.1. Coupling of PEG-alkene / -alkyne by photoreaction to partially reduced antibody
Photochemical conditions have been developed for the coupling of antibodies to PEG-linker (F) and (G) alkene / alkyne functional groups. Therefore, various reaction conditions such as light exposure time, PEG excess, photoinitiator I (“Photo I”) concentration, protein concentration and reaction pH value should be investigated. Coupling efficiency was analyzed by SDS-PAGE of the obtained conjugate.

4.2 (Partial) Reduction of Experimental Antibodies TCEP was added to an antibody solution with a protein concentration of -15 mg at a final concentration of 0.01-1 mM, which corresponds to a 0.1-10-fold molar excess compared to the antibody. The reduction reaction was carried out at room temperature for 1 hour in 20 mM sodium phosphate, pH 7.2. Subsequently, the antibody was modified with PEG-alkene or PEG-alkyne by photo click chemistry.
Modification of Antibodies with PEG-Alkene / -Alkyne by Optical Click Chemistry-With PEG-Alkene Lot CES0601 or PEG-Alkin Lot CES0602 by photoinduction using caproBox ™ (Caprotech) under the various conditions described in the sections below. The antibody was modified.

4.3. Results
A commercially available antibody IgG1 (mouse) was used for the initial photochemical coupling experiment of PEG-alkene. The antibody was reduced with 1 mM TCEP (10 eq) at a protein concentration of 15 mg / mL for 1 hour. The effect of excess PEG on antibody modification was tested with reduced IgG1 under the following conditions.
・ IgG1 concentration: 1 mg / mL
-Reaction buffer: 20 mM Na phosphate, pH 7.2
・ Mole excess of PEG-alkene (F) compared to IgG1: 0.2 to 10 times ・ Photoinitiator I excess: 0.1 mol / 1 mol of PEG
・ Light exposure time: 30 minutes
PEGylation of IgG1 was monitored by SDS-PAGE analysis.

SDS-PAGE detected two bands at 55 kDa and 25 kDa for reduced but non-PEGylated IgG1 and attributed to the heavy and light chains, respectively. Several high molecular weight bands were observed for all samples bound to the PEG-alkene by photoinduction. These bands are inconsistent with the expected molecular weight difference of about 10 kDa for the PEGylated antibody fragment and thus almost certainly correspond to aggregated proteins or misconstructed proteins. Bands are observed at about 65 kDa only with 10 equivalents of PEG excess and are attributed to monoPEGylated heavy chains. No additional bands were detected in IgG1 samples containing PEG and photoinitiator but not exposed to light, except for heavy and light chains. This suggests that light exposure caused agglomeration.

Since the PEGylated heavy chain was obtained, modification of the antibody by optical click chemistry was assumed to be feasible, but the reaction had to be optimized for the reduction of agglutinating antibodies.
Various light exposure times and photoinitiator I concentrations were investigated to reduce protein aggregation and increase PEGylation. Therefore, TCEP-reduced IgG1 was modified with PEG-alkene using the following conditions.
・ IgG1 concentration: 1 mg / mL
-Reaction buffer: 20 mM Na phosphate, pH 7.2
・ Mole excess of PEG-alkene compared to IgG1: 10 times ・ Photoinitiator I excess: 1-10 mol / 1 mol of PEG
・ Light exposure time: 5 seconds to 10 minutes

Increased photoinitiator I excess resulted in a higher fraction of aggregation at all light exposure times, but the proportion of monoPEGylated heavy chains was unaffected. Aggregate fractions could be significantly reduced by reducing light exposure to 5-30 seconds, but even within this time range, monoPEGylated heavy chain fractions decreased with exposure time.
To minimize the possible misconstitution of interchain disulfide formation, we decided to reduce the excess of TCEP to 1 or 5 equivalents compared to IgG1 during initial antibody reduction. The antibody was then modified with 1 equivalent of PEG-alkene with 1 equivalent of photoinitiator I at different photoexposure times.
By reducing the TCEP excess, one equivalent of TCEP was able to reduce the aggregate ratio to nearly 9% with a 10 minute light exposure time. However, short exposures are strongly required for the final chip coating process, resulting in a 13% monoPEGylated heavy chain with a 1x TCEP excess and a 15% monoPEGylated heavy chain with a 5x TCEP excess. I decided to choose a standard time of 0.5 minutes. In addition, about 1% and 3% monoPEGylated light chains were detected with 1 and 5 equivalents of TCEP, respectively. A significantly lower percentage of aggregates was detected with 1 equivalent of TCEP, so we decided to select it as a standard TCEP excess.
The PEG excess was further optimized for standard light exposure time using the following conditions.
・ IgG1 concentration: 1 mg / mL
-Reaction buffer: 20 mM Na phosphate, pH 7.2
・ Mole excess of PEG-alkene compared to IgG1: 0.5 to 10 times ・ Photoinitiator I excess: 1 mol / 1 mol of PEG
・ Light exposure time: 30 seconds
A higher proportion of monoPEGylated heavy chains was observed with increasing PEG excess.

The highest proportion (7%) monoPEGylated heavy chain was obtained with 10 equivalents of PEG and was selected as the standard PEG excess.
The protein concentration in the reaction mixture was optimized using the following reaction parameters.
・ IgG1 concentration: 0.2-10mg / mL
-Reaction buffer: 20 mM Na phosphate, pH 7.2
・ Mole excess of PEG-alkene compared to IgG1: 10 times ・ Photoinitiator I excess: 1 mol / 1 mol of PEG
-Light exposure time: 30 seconds The proportion of mono-PEGylated heavy chains increased as the protein concentration increased. However, the proportion of high molecular weight impurities (aggregates and PEGylated antibodies) also increased.
The highest proportion (16%) of mono-PEGylated heavy chains was detected at a protein concentration of 2 mg / mL. However, in order to obtain a high protein concentration, the antibody must be concentrated in the previous step. Due to the strong need for chip coating, antibody, antibody concentration and PEG: protein ratio will have to be further optimized. Therefore, for reactions in solution, a standard protein concentration of 1 mg / mL was selected to minimize preconcentration of the antibody.

The reaction pH value was optimized using the following reaction parameters.
・ IgG1 concentration: 1 mg / mL
-Reaction buffer and pH value: 100 mM Na acetate, pH 5.0; 20 mM Na phosphate, pH 7.2; 100 mM Na borate, pH 9.0
・ Mole excess of PEG-alkene compared to IgG1: 10 times ・ Photoinitiator I excess: 1 mol / 1 mol of PEG
-Light exposure time: 30 seconds At reaction pH 5, the highest proportion (2%) of monoPEGylated light chains was detected, but the lowest proportion (6%) of monoPEGylated heavy chains was obtained. A slightly higher proportion (7%) of mono-PEGylated light chain was obtained at pH 7. At pH 9, the highest percentage (9%) of monoPEGylation was detected, but the highest percentage (22%) of HMWI was also obtained. Therefore, we decided to use a standard reaction pH value of 7 where a small (18%) HMWI was detected.
The following standard reaction parameters were selected.
・ Protein concentration: 1 mg / mL
-Reaction buffer: 20 mM Na phosphate, pH 7.2
・ Mole excess of PEG-alkene / -alkyne compared to IgG1: 10 times ・ Photoinitiator I excess: 1 mol / 1 mol of PEG
・ Light exposure time: 30 seconds

Standard reaction parameters were introduced into the modification of PEG-alkyne and anti-ACTH antibodies. Since reduction of IgG1 and anti-ACTH antibody was achieved with various reducing agents in the previous experiment, various TCEP concentrations were investigated.
The highest percentage (about 7%) of monoPEGylated protein (using PEG-alkene or PEG-alkyne) for both conjugates was obtained with a minimum TCEP excess (0.1 eq), which was chosen as the standard condition. ..
According to non-reducing SDS-PAGE, PEGylation at the cysteine residue of the anti-ACTH antibody did not affect the quaternary structure of the antibody. On the reducing SDS-PAGE of the conjugate, bands were detected at 66 and 75 kDa and assigned to mono-PEGylated heavy chains and di-PEGylated heavy chains, respectively. No PEGylated light chain was observed by SDS-PAGE.

Introduction of thiol groups with S-acetylthioacetic acid N-succinimidyl (SATA) followed by modification with PEG-alkene
According to SEC analysis, the quaternary structure of the antibody was unaffected by partial reduction and binding of PEG to the respective cysteine residues. However, reduction of disulfide bonds can weaken antibody stability and interfere with its activity. Therefore, the introduction of free thiol groups using the bifunctional reagent SATA should be investigated. By this technique, SATA is first attached by its NHS functional group to the free amino group of the lysine residue in the antibody. The conjugated sulfhydryl group of the SATA is then deprotected by treatment with hydroxylamine.

The resulting free thiol group should be attached to the PEG-alkene / -alkyne by photochemical under the conditions previously developed.
5.1. Experimental Antibodies SATA Adhesion to Free Amino Groups-SATA and Sulfhydryl Addition Kits (Thermo Scientific) were used for SATA attachment to IgG1 and anti-ACTH antibodies. The reaction was carried out according to the manufacturer's instructions. Antibodies at a concentration of 1 mg / mL in 20 mM sodium phosphate, pH 7.2 were modified with SATA in 1, 3, 5 and 10 eq with a molar excess. The reaction was carried out at room temperature for 2 hours. The conjugated sulfhydryl group of SATA was deprotected by treatment with hydroxylamine amine at a concentration of 5 mg / mL for 2 hours at room temperature. Residual free SATA and hydroxylamine were removed using a CentriSpin 10 column.
Binding of PEG-alkene / -alkyne to free SATA thiol group-PEG-alkene / -alkyne attached to antibody-bound SATA free thiol group by photoinduction using caproBox ™ (Caprotech) rice field. This reaction was carried out at 20 mM sodium phosphate, pH 7.2 with a protein concentration of 1 mg / mL and a 10-fold excess of PEG-alkene / -alkyne. 1 mol of photoinitiator I per mol of PEG was added and a light exposure time of 30 seconds was selected.

5.2. Results In the initial experiment, the attachment of PEG-alkene to the free thiol group of the SATA molecule attached to the amino group of antibody IgG1 was investigated using different SATA excesses. The resulting PEGylated reaction mixture was analyzed by SDS-PAGE.
In non-reducing SDS-PAGE, the main band was detected at 155 kDa corresponding to complete IgG1. Further bands with lower molecular weights (about 135 and 110 kDa) were assigned to partially dissociated IgG1. The proportion of these bands increased with increasing SATA excess. This suggests that SATA attachment may have affected the quaternary structure of the antibody. Therefore, the excess of SATA compared to the antibody should be minimized. On reducing SDS-PAGE, a 66 kDa band, up to 1-2% of the total protein, corresponded to a monoPEGylated heavy chain. Further bands of higher molecular weight were assigned to high molecular weight impurities (HMWI). These may correspond to aggregates or multiple PEGylated proteins. For further investigation, a control reaction was performed in which IgG1 was modified for it with 3 or 10 equivalents of SATA. Then, a photoclick reaction was carried out under standard conditions without adding PEG-alkene.
In reducing SDS-PAGE, the band indicating HMWI observed in the PEGylation reaction was detected. No PEG was added to the mixture during the photoclick reaction, so these bands were assigned to the aggregated protein.
Subsequent experiments examined the attachment of PEG-alkene to SATA bound to anti-ACTH antibody in 1- and 3-fold excesses by optical click chemistry.
When the anti-ACTH antibody was used, a lower proportion of aggregates was observed after the photoclick reaction compared to IgG1. The band corresponding to the monoPEGylated heavy chain of the anti-ACTH antibody was detected by reducing SDS-PAGE analysis. Therefore, PEGylation of the antibody was considered feasible, but the proportion of monoPEG-HC was low (<1%).

Surface modification of gold surface with alkene / alkyne and covalent protein (antibody) via photochemical
6.1. Experiment The equipment listed in the table below is an example. Equipment of comparable quality from other manufacturers may be used instead.

6.2. Materials The materials listed in the table below are examples. Materials of comparable quality from other manufacturers may be used instead.

6.3. Preparation of solution and buffer ○ 10 mM sodium phosphate buffer, pH 7.2
Weigh 710 mg of disodium hydrogen phosphate in a 250 mL beaker and fill the beaker with 250 mL of ddH ₂ O; weigh 780 mg of sodium dihydrogen phosphate into another 250 mL beaker and fill the beaker with ddH ₂ O. ..
-Put 250 mL of disodium hydrogen phosphate in a 500 mL beaker and titrate with dihydrogen phosphate until pH 7.2 is reached.
• Filter the solution using a 0.2 μm polyether sulfone membrane filter and place in a 500 mL glass bottle.
-Store the solution at room temperature for up to 6 months.

○ 25 mg / mL TCEP stock solution in dd H ₂ O ・ Weigh 25 mg of TCEP into a 1.5 mL screw lid tube.
-Add 1 mL of ddH ₂ O.
-Vortex the tube until TCEP is completely dissolved.
・ Store at 4 ℃.
○ 20 mM sodium phosphate buffer, 1 mg / mL photoinitiator in pH 7.2 ・ Weigh 10 mg of photoinitiator into a 15 mL screw lid tube.
-Add 10 mL of 10 mM sodium phosphate buffer, pH 7.2.
-Shake the solution at 40 ° C for 10 minutes to dissolve the photoinitiator.
-Pass the solution through a 0.2 μm polyether sulfone syringe filter.
-Prepare the solution on the day of use.

○ 10 mM PEG-alkene / -alkyne solution and gold surface modification in DMAC ・ Store PEG-alkene / -alkyne at -20 ° C; thaw at room temperature for about 30 minutes.
-Weigh an appropriate mass of PEG-alkene / -alkyne into a 15 mL screw lid tube (5000 Da PEG-alkene → dissolve 500 mg in 1 mL DMAC to obtain 100 mM stock solution).
-Vortex the PEG solution until the PEG is completely dissolved.
-A 100 mM stock solution can be divided into equal parts and stored at -20 ° C.
-As a result, prepare a working solution on the day of use:
-Dilute the stock solution with DMAC to a working solution concentration of 10 mM.
-Invert the solution and vortex to homogenize.
• In a draft, pipette a 10 μL 10 mM PEG-alkene / alkyne solution onto a purified (cleaning protocol) gold CMOS surface and react in a glass Petri dish at 20 ° C for 60 minutes.
• Clean the gold CMOS surface with ddH ₂ O for 10 seconds in a 250 mL beaker.
-Dry the gold CMOS surface with compressed air.
・ Dry storage at 20 ° C until use.

○ TCEP reduction of disulfide bonds ・ Put 20 μL of antibody (0.2 mg / mL, MW = 150 kDa) into a 0.5 mL cup with a pipette.
• The TCEP stock solution is diluted 1: 100 and 1: 2 twice with sodium phosphate buffer to give a final TCEP concentration of 3 x molar excess with respect to antibody concentration.
-Add 20 μL of TCEP to the antibody.
-Incubate the reduction mixture at room temperature and stirring at 30 rpm for 60 minutes.
○ Put 15 μL of spotting buffer into the Genetix Plate with an antibody spotting and coupling pipette.
-Add 2.24 μL of photoinitiator stock solution.
-Add 12.76 μL of reducing antibody solution to make the concentration twice higher than the spotting concentration.
・ Spotting (standard spotting procedure)
-Place the UV lamp in a spotting device and expose it to UV light (λ = 302 nm) at a distance of 10 mm above the gold surface for at least 4 to 10 minutes.
・ Wash with ddH ₂ O and dry with compressed air.
-Block the surface with the desired blocking agent (blocking protocol).
・ Wash with ddH ₂ O and dry with compressed air.
-Use CMOS for the intended assay.

The upper part of FIG. 4 is a metal surface (1) modified with a crosslinker molecule (4) covalently bonded to the metal surface via a dithiol group, which has a polyethylene glycol (PEG) group as a spacer and a terminal allyl group. ) Is schematically shown. When a biomolecule with at least one sulfhydryl group (2), photoinitiator and UV light irradiation is applied, the biomolecule is immobilized on the surface via a crosslinker, as shown in the lower part of FIG. 4 (3). Will be done.

Examples 7-9
The results of different immobilization experiments made to evaluate the surface modification of CMOS chips are shown in the fluorescent photographs of Figures 1-3.
The CMOS chip contains 128 electrodes, each of which can be addressed by spotting (compare Example 6). The corresponding spotting layout is also shown.

Effect of UV light and photoinitiator compared to lipoamide-PEG (11) -maleimide modified surface.
In the experiment below, a fluorescence photograph was taken from a CMOS chip consisting of 128 gold electrodes as the sensor surface. Different dilutions and spotting buffers were used for spotting the polyclonal rabbit anti-ACTH antibody. ACTH was then added to the surface and a complete assay for ACTH was performed. The monoclonal mouse anti-ACTH antibody binds to the bound ACTH after addition, which can be visualized with a fluorescently labeled rabbit anti-mouse antibody.
Fluorescence intensity is directly proportional to the amount of associated polyclonal rabbit anti-ACTH antibody. FIG. 1 shows the different conditions of photoreaction compared to the lipoamide-PEG (11) -maleimide modified surface.
A: Lipoamide-PEG (11) -maleimide modified surface with 10 minutes UV irradiation at a wavelength of 304 nm. Only the presence of weak fluorescence can be observed. This means that the antibody immobilization reaction occurred only to a low degree.
B: R-α-lipoic acid-PEG12-propargyl modified surface of the present invention with an irradiation time of 7.5 minutes. It is clear that antibody immobilization must have occurred due to the higher fluorescence intensity.
It should be noted that samples KIA5 to KIA7 did not use photoinitiators, whereas samples KIA1 to KIA4 were produced with photoinitiators. All antibodies were pretreated with TCEP and different excesses of TCEP (30 x, 3 x molar excess relative to antibody concentration) were utilized.
C: R-α-lipoic acid-PEG12-propargyl modified surface without UV irradiation. It is clear that without UV irradiation, only slight immobilization of the antibody occurs on the surface.
D: Spotting layout KIA represents different reaction conditions for the polyclonal rabbit anti-ACTH antibody.
SPO: Represents a spotting control to which only spotting buffer is applied.

Immobilization of Monoclonal Mouse Anti-ACTH Antibodies with and without Photoinitiator This experiment demonstrated the adaptability of the photochemicals to the immobilization of monoclonal mouse antibodies. The antibody was directly stained with a fluorescently labeled anti-mouse antibody. The corresponding results are shown in Figure 2.
FIG. 2 shows immobilization of a monoclonal mouse antibody on an R-α-lipoic acid-PEG12-propargyl modified surface according to the present invention with the help of photoreaction and irradiation time of 4 minutes.
A: Shows a comparison of immobilization efficiency with and without the use of photoinitiator. It is clear that the use of photoinitiators provides strong and uniform immobilization of the monoclonal antibody. However, without the photoinitiator, the immobilization efficiency is less clear and zone formation is observed. Nevertheless, it can be assumed that the reaction can be carried out in a sufficient irradiation time without a photoinitiator.
B: Spotting layout KIA corresponds to a polyclonal rabbit anti-ACTH antibody that cannot be stained.
SPO: Represents a spotting control to which only spotting buffer is applied.
MIA: Represents a monoclonal mouse antibody in which MIA1 and MIA2 are produced with or without a photoinitiator. All antibodies were previously treated with TCEP.

Comparison of allyl and propargyl functionality and regular and non-regular PEG chain lengths.
In this experiment, the reactivity of allyl and propargyl groups was compared. Furthermore, the difference in reactivity between the specified and non-specified PEG chain lengths was investigated. The unspecified PEG chain length has a molecular weight of 5 kDa. Assays were performed using ACTH as the analyte as described in Example 7. The CMOS chips used were pretreated with a benzocyclobutene (BCB) layer in which the gaps between the electrodes showed fluorescence enhancement on it. Antibodies were applied within a 1: 2 dilution series starting at 100 μg / mL. The corresponding results are shown in Figure 3. The irradiation time was 4 minutes.
FIG. 3 shows the immobilization of the polyclonal rabbit anti-ACTH antibody by the photoreaction of the present invention on different modified surfaces.
A: R-α-lipoic acid-5kDa PEG-propargyl (Example 1.5) modified surface.
B: R-α-lipoic acid-PEG12-propargyl (Example 1.2) modified surface.
C: R-α-lipoic acid-5kDa PEG-allyl (Example 1.4) modified surface.
D: R-α-lipoic acid-PEG12-allyl (Example 1.1) Modified surface.
E: Spotting layout KIA corresponds to the polyclonal rabbit anti-ACTH antibody, which has the highest concentration of KIA (100 μg / mL as a spotting solution) while KIA4 has the lowest concentration (12.5 μg / mL as a spotting solution). It is clear that D exhibits the highest intensity of fluorescence. All other modified surfaces show slightly lower strength, but specific immobilization can also be observed here.

Claims (13)

A method for immobilizing a biomolecule containing at least one sulfhydryl group, wherein the following steps:
(a) If necessary, a step of treating the biomolecule with a reducing agent to cleave existing-SS-bonds in the biomolecule, or
(b) If necessary, a step of treating the biomolecule with an acylating agent having a protected sulfhydryl group to deprotect the sulfhydryl group;
(c) Step of contacting the surface of the modified metal with the biomolecule;
(d) Including the step of irradiating the resulting surface with UV in the presence of a photoinitiator.
The metal surface is
The method described above, which is modified with the cross-linker compound of the following formula (I):
HS- (CH ₂ ) _m -CH (ZH) -SPACER- (CH ₂ ) _p -A (I)
(During the ceremony,
m is an integer from 2 to 6
A is selected from -CH = CH ₂ and -C≡CH, and
Z is S or a single bond,
SPACER is the following formula
-(CH ₂ ) _n- (C = O) _x -Y-(CH ₂ CH ₂ -O) _y- (CH ₂ ) _r- (C = O) _v -X-
Is the basis of
here,
X and Y are independently NH or O, respectively.
n is 0 or an integer from 1 to 10 and
x and v are 0 or 1 independently, respectively.
y is an integer from 1 to 20
r and p are independently selected from integers 1-6). In the cross-linker compound of formula (I)
Z is S,
A is CH = CH ₂ and
m is an integer from 2 to 4,
X and Y are NH,
n is 0 or an integer from 1 to 10 and
x and v are 1
y is an integer from 1 to 20
r and p are 1,
The method according to claim 1. The method of claim 1 or 2, wherein the biomolecule is an antibody, enzyme or nucleic acid. The photoinitiator is a 1-benzoyl-1-methyl-ethanol derivative,
The method according to any one of claims 1 to 3. Irradiation by UV irradiation is performed at a wavelength λ _max of 300 to 340 nm, preferably about 320 nm.
The method according to any one of claims 1 to 4. The following formula (I)
HS- (CH ₂ ) _m -CH (ZH) -SPACER- (CH ₂ ) _p -A (I)
(During the ceremony,
m is an integer from 2 to 6
A is selected from -CH = CH ₂ and -C≡CH, and
Z is S or a single bond,
SPACER is the following formula
-(CH ₂ ) _n- (C = O) _x -Y-(CH ₂ CH ₂ -O) _y- (CH ₂ ) _r- (C = O) _v -X-
Is the basis of
here,
X and Y are independently NH or O, respectively.
n is 0 or an integer from 1 to 10 and
x and v are 0 or 1 independently, respectively.
y is an integer from 1 to 20
r and p are independently selected from integers 1 to 6)
Cross-linker compound. The following formula (IA)
HS- (CH ₂ ) _m -CH (SH) -SPACER- (CH ₂ ) _p -A (IA)
(In the formula, A, SPACER, m and p have the meaning given to the formula (I) of claim 6).
Cross-linker compound. The cross-linker compound of the formula (IA) according to claim 7.
m is an integer from 2 to 4,
p is 1
SPACER is the following formula
-(CH ₂ ) _n- (C = O) -NH-(CH ₂ CH ₂ -O) _y -CH _2- (C = O) -NH-
(During the ceremony,
n is 0 or an integer from 1 to 10 and
y is an integer from 1 to 20)
The cross-linker compound which is the basis of. The following formula (II)

(In the formula, A, SPACER, m and p have the meaning given to the formula (I) of claim 6).
The compound for synthesizing the cross-linker compound of the formula (I) according to claim 6 by reduction . A modified metal surface in which at least one thiol group of the compound of the formula (I) or (IA) according to any one of claims 6 to 8 is covalently bonded to at least one of the metal atoms on the surface. The modified metal surface. The modified metal surface of claim 10, wherein the metal is gold . A kit for performing the biomolecule immobilization method according to any one of claims 1 to 5, which is as follows.
(vi) The substrate having the modified metal surface according to claim 10 .
(vii) Optional storage unit containing the appropriate reducing agent, or
(iii) An optional storage unit containing a suitable acylating agent having a protected sulfhydryl group and a deprotecting agent for the sulfhydryl group.
(ix) Storage unit containing the appropriate photoinitiator, and
(v) The kit comprising a pamphlet explaining the conditions for performing this method. A kit for performing the biomolecule immobilization method according to claim 12, further comprising a device suitable for UV irradiation.

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